Method for In Vivo Expansion of T Regulatory Cells

ABSTRACT

Compositions specific for TNF-receptor superfamily member 25 (TNFRSF25, DR3) modulate the immune response by regulating T regulatory cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.61/273,299, filed Aug. 3, 2009, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under grant numbers5PO1CA109094-03 awarded by the National Cancer Institute and5RO1AI061807-05 awarded by the National Institute of Allergy andInfectious Diseases. The U.S. government has certain rights in theinvention.

FIELD OF THE INVENTION

Embodiments of the invention relate to compositions and methods forregulating T cells in vivo. In particular, the compositions and methodsregulate human CD4⁺FoxP3⁺ cells.

BACKGROUND

The tumor necrosis factor superfamily (TNFSF) consists of at least 19ligands and 30 receptors (TNFRSF) that are differentially and temporallyexpressed by both lymphoid and non-lymphoid cells. In CD3⁺ T cells,TNFSF signals function in both antigen specific and non-specific ways tosupport various phases of an immune response including polarization,expansion, effector function, contraction, memory and death. TNFSF15(TL1A) is the ligand for TNFRSF25 (DR3, hereafter referred to as TNFR25)and can modulate TNFR25-expressing T and NKT cells either positively ornegatively by triggering TRADD or FADD signaling cascades via the deathdomain-containing cytoplasmic tail of TNFR25. TNFR25 signaling is animportant contributor to the pathology observed in a range ofauto-inflammatory conditions including asthma, inflammatory boweldisease (IBD), experimental autoimmune encephalomyelitis (EAE) andrheumatoid arthritis (RA). Antibody blockade of TL1A can prevent acuteasthma in mice and genetic knockout of TNFR25 significantly blunts thepathologic events in experimental models of EAE or RA. TL1A contributesto the development of these disease by enhancing polarization,differentiation and effector function of NKT, Th2 and Th17 cells.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

Signaling through the TNF-receptor superfamily member 25 (TNFRSF25, DR3)on CD4⁺ T cells, where it is constitutively expressed, enhances T_(H)2and T_(H)17 cytokine production and contributes to pathologicalinflammation in disease models of asthma, inflammatory bowel disease,multiple sclerosis (MS), experimental autoimmune encephalitis andrheumatoid arthritis.

In preferred embodiments, agents which modulate TNFRSF25 signalingmodulate immune cell response. These agents provide novel therapies fordiseases and conditions thereof. For example, an agonist of TNFRSF25 ledto the rapid and extensive in vivo expansion of CD4⁺FoxP3⁺ cells to30-35% of all CD4⁺ cells within four days of administration. The rise inCD4⁺FoxP3⁺ cells was due to increased proliferation of CD4⁺FoxP3⁺CD25⁺cells expressing high levels of GITR and CD103. TNFRSF25 agonistexpanded CD4⁺FoxP3⁺ cells retained TGF-β-dependent suppressive activityex vivo, which however was susceptible to abrogation by continuedTNFRSF25 signaling. TNFRSF25 signaling in addition to modulatingeffector cell responses plays an important role in both the inductionand resolution of inflammatory responses via control of T regulatorycell expansion and activity.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: show that TNFR25 stimulates the rapid proliferation ofCD4+FoxP3+ cells in vivo. FIG. 1A: Differential expression of TNFR25,GITR, OX40 and 4-1BB on conventional and regulatory T cells. TNFRSFexpression was determined by flow cytometry on highly purifiedCD4⁺FoxP3⁻ (Tconv) and CD4⁺FoxP3⁺ Treg from splenocytes harvested fromuntreated FIR mice. FIG. 1B: Kinetics and dose-dependent expansion ofCD4⁺FoxP3⁺ Treg cells in peripheral blood after 4C12 injection.FoxP3-RFP reporter (FIR) mice were injected intraperitoneally (i.p.)with the amount of purified 4C12 indicated. The mice were bled daily andFoxP3-RFP expression analyzed in peripheral blood cells by flowcytometry. FIG. 1C: Treg expansion was compared following treatment withother TNFR agonistic antibodies. Mice were injected i.p. with theindicated antibodies (100 μg) on day 0. Mice were bled daily as in FIG.1B for 6 days, the percentage of peripheral blood Treg out of total CD4⁺T cells on day 4 is shown. These data have been reproduced in over 8independent experiments. Error bars indicate mean±SEM. Significance wasdetermined by the student's t-test (FIG. 1B) or one-way ANOVA with Tukeypost test (FIG. 1C). * indicates p<0.05, ** indicates p<0.01, ***indicates p<0.001.

FIGS. 2A-2F show that TNFR25 induced Treg expansion requires TCR andIL-2 signaling. CD4⁺ cells were highly purified by FACS sorting from FIRmice and adoptively transferred into MHCII^(−/−) or CD4^(−/−) mice.Following adoptive transfer, recipient mice were treated with either4C12 or isotype control antibody and the percentage (FIG. 2A) andabsolute number (FIG. 2B) of FoxP3-RFP positive cells was analyzed 4days after antibody treatment. FIR mice were treated with cyclosporin-A(FIG. 2C) or FK506 (FIG. 2D) or a vehicle control from day-1 throughday-4 by i.p. injection as described in the methods. Mice were treatedwith either 4C12 antibody or IgG control antibody and the proportion ofFoxP3-RFP positive cells relative to total CD4⁺ cells in the peripheralblood was analyzed on day 4. IL-2 receptor beta deficient mice (FIG. 2E)or CD80/86^(−/−) mice (FIG. 2F) were analyzed for the proportion ofCD4⁺FoxP3⁺ cells out of total CD4⁺ splenocytes 4 days after treatmentwith either 4C12 or isotype control antibody, as compared to C57BL/6control mice. These data are represented as the mean±S.E.M. of at least2 independent experiments with ≧3 mice per group per experiment.**indicates p<0.01, ***indicates p<0.001.

FIGS. 3A-3E show that TNFR25 expanded Treg are hyper-responsive to IL-2and require Akt activation. FIG. 3A: CD4+FoxP3+ cells were purified fromFIR mice on day 4 after treatment with control IgG or 4C12 antibodiesand incubated with the indicated amounts of IL-2 in vitro. CD4⁺FoxP3⁺cell proliferation was measured on day 3 of the culture by incorporationof tritiated thymidine. FIG. 3B: CD4⁺FoxP3⁺ cells were purified as in(FIG. 3A) and the surface expression of IL-2Rγ (CD132) or IL-2Rβ (CD122)was determined by flow cytometry. FIG. 3C: CD4⁺FoxP3⁺ cells purifiedfrom FIR mice 4 days after treatment with IgG or 4C12 antibody wereanalyzed for expression of pSTAT5 15 minutes after treatment with 10ng/ml of IL-2 in vitro. FIR mice were treated once daily with rapamycin(FIG. 3D) or twice daily with Akt inhibitor V (FIG. 3E) or a vehiclecontrol from day-1 through day-4 by i.p. injection as described in themethods. Mice were treated with either 4C12 or control IgG antibody onday 0 and the proportion of FoxP3-RFP positive cells relative to totalCD4⁺ cells in the peripheral blood was analyzed on day-4. These data arerepresented as the mean±S.E.M. of at least 2 independent experimentswith ≧2 mice per group per experiment. ns indicates not significant, ***indicates p<0.001.

FIGS. 4A-4E show that in vivo Treg expansion by TNFR25 inhibitsinflammation in allergic asthma. Allergic asthma was induced byimmunization with ova/alum followed by aerosol challenge with ova/PBS asdescribed in materials and methods. FIG. 4A: Peripheral blood wascollected and analyzed for the fraction of CD4⁺FoxP3⁺ cells out of totalCD4⁺ T cells from ova/alum immunized mice as compared to non-immunizedmice following treatment with either 4C12 or isotype control antibody.Data indicate mean±SEM. FIG. 4B: Total lung cells were harvested andanalyzed by flow cytometry. The total number of each indicated cellpopulation are shown. Data indicate mean±SEM. FIG. 4C: The percentage ofCD4⁺FoxP3⁺ Treg out of total CD4⁺ T cells. FIG. 4D: Bronchio alveolarlavage fluid (BALF) was collected 3 days after aerosolization withova/PBS as described. The total number of eosinophils is shown. Dataindicate mean±SEM. FIG. 4E: Total RNA was extracted from total lungcells and used for real-time RT-PCR. The expression levels of IL-4, IL-5and IL-13 in 4C12 or isotype control treated mice are shown relative tosaline-aerosolized control lung cells. FIG. 4F: Lungs were harvested andsectioned for histological sections. H&E (left panels) as well as PAS(right panels) were obtained for each treatment group. Representativeimages are shown. These data have been repeated in four independentexperiments with at least 3 mice/group/experiment. FIG. 4G: PAS stainedsections were quantitated using Image J software as described in themethods. Two representative images were quantitated from each of ≧5 micefrom 2 separate experiments. Statistical significance was determined byone-way ANOVA with Tukey's post-test. * indicates p<0.05, ** indicatesp<0.01, *** indicates p<0.001 of either 4C12 or IAC as compared to theIgG group or saline-control group, as indicated.

FIGS. 5A-5F show that TNFR25 stimulation leads to Treg expansion in vivoby inducing proliferation of existing CD4⁺FoxP3⁺ CD25^(int) cells. FIG.5A: FIR mice were treated with IgG or 4C12 on day 0 and splenocytes wereharvested 4 days later and analyzed by flow cytometry. Representativeflow cytometry plots from peripheral blood cells collected from mice 4days after the indicated treatment. FIG. 5B: The average ratio ofCD25^(hi) versus CD25^(int) Treg in splenocytes 4 days after theindicated treatment. FIG. 5C: Representative dot plots are shown thatwere pre-gated on CD4⁺, FoxP3⁺ cells. Percentages indicate thecontribution of each phenotype toward the total fraction of CD4⁺FoxP3⁺cells. FIG. 5D: The average proportion of Ki67⁺ or Ki6T among CD25^(hi)and CD25^(int) Treg following the indicated treatment as described inFIG. 5A is shown. Data are mean±SEM. FIG. 5E: CD4⁺FoxP3⁻ and FIG. 5F:CD4⁺FoxP3⁺ cells were sorted from CD45.2⁺ FIR mice to >99% purity and2×10⁶ cells from each subset were adoptively transferred into CD45.1congenic B6-SJL mice. 24 h later, mice were injected i.p. with 20 mg4C12 or IgG, respectively. FIGS. 5E, 12G: Transfer of CD4^(−/−) FoxP3⁻and (FIGS. 5F, 5H) CD4⁺FoxP3⁺ cells into congenic CD45.1 B6-SJL mice.FIGS. 5E, 5F: Histogram showing the percentage of CD45.2⁺ and RFP(FoxP3⁺) cells among CD4⁺ cells on day 5 after adoptive transfer. FIGS.5G, 5H: Kinetics of transferred cell contraction following 4C12 orhamster IgG treatment. The percentage of transferred cells (CD45.2⁺CD4⁺)out of host CD45.1⁺CD4^(−/−) FoxP⁺ cells (FIG. 5E) or CD45.2⁺CD4⁺FoxP3⁺out of host CD45.1⁺CD4⁺ cells (FIG. 5F) are shown. Error bars indicateaverage percentage±SEM for 3 mice per group for each of two independentexperiments. * indicates p<0.05 and ** indicates p<0.01.

FIGS. 6A-6F show the suppressive activity of in vivo expanded Treg.CD4⁺FoxP3⁺ Treg cells were sorted from 4C12 and IgG isotype controlinjected mice on day 4 and subjected to a standard in vitro suppressionassay using CD4^(−/−) FoxP3⁻CD25⁻ cells as Tconv and soluble α-CD3 (2μg/ml) for 72 h (96-well, round bottom plate). The assay was performedin the absence (FIGS. 6A, 6C) or presence (FIG. 6B, 6D) of 1:1 antigenpresenting cells (APCs) using different ratios of Treg:Tconv (FIGS. 6A,6B). In FIGS. 6C and 6D, IgG, 4C12 or DTA1 (10 g/ml) antibodies wereadded to the suppression assay. The Treg:Teff ratio was kept constant ata 1:2 ratio. FIG. 6E: Teff cells from TNFR25 dominant negative (DN) miceand Treg cells from wt mice were used. IgG or 4C12 antibodies (10 μg/ml)were added to the suppression assay. The Treg:Teff ratio is 1:2. FIG.6F: CD4⁺CD25^(hi) and CD4⁺CD25^(int) Treg cells from IgG or 4C12injected mice were used. ³H-thymidine was added for the last 6 h beforethe assay was analyzed on a scintillation counter. Percent proliferationwas calculated using the counts obtained for the indicated condition asa percentage of the total counts obtained in wells containing Teff inthe absence of Tregs. Data are expressed as the average±SEM with ≧4samples for each condition in each of two independent experimentswith >6 mice per group per experiment.

FIGS. 7A, 7B show the comparison of Treg expanded by treatment with 4C12or recombinant IL-2/anti-IL-2 antibody complex (IAC). FIG. 7A: FIR micewere treated with 4C12 (10 μg) on day 0 or with a series of threeinjections with IAC on days 0-2. The proportion of FoxP3⁺ cells withinthe CD4+ T cell population was measured in the peripheral blood daily byflow cytometry. FIG. 7B: Splenocytes were isolated from FIR mice 4 onday 4 after treatment with IAC, 4C12 or isotype control IgG. Theproportion of CD4⁺FoxP3⁺ cells expressing CD25 and the proliferationmarker Ki67 are shown.

FIGS. 8A-8D show that 4C12 treatment induces Treg expansion in alltissues analyzed. FIG. 8A: An example of a typical flow cytometry dotplot staining for CD4 and FoxP3 (RFP). CD4⁺FoxP3⁺ cells from quadrantQ2-1 were gated for subsequent analysis of CD25^(hi) and CD25^(int)cells as shown in FIGS. 8B-8D. FIG. 8B: The ratio of GITR and FIG. 8C:CD103 expression among CD25^(hi) versus CD25^(int) Treg in splenocytes 4days after the indicated treatment. FIG. 8D: Data are represented asmean±SEM from over 8 independent experiments with at least 3 mice pergroup per experiment. Paired analysis was performed using the studentsT-test. ** indicates p<0.01 and *** indicates p<0.001.

FIGS. 9A-9B show an example of sorting strategy and the resultsobtained. FIG. 9A: Splenocytes were harvested from FIR mice, enrichedfor CD4⁺ T cells and sorted on the basis of CD4⁺ and FoxP3⁺ (RFP). Theleft panel illustrates a typical CD4-enriched population of splenocytes.The middle and right panels illustrate representative post-sort analysisfor CD4⁺FoxP3⁺ (P3 gate) and CD4⁺FoxP3⁺ (P4 gate) populations. FIG. 9B:For some experiments CD4⁺FoxP3⁺ cells (gate P3) were sorted based onCD25 expression. Representative plots are shown demonstrating the gatingstrategy for CD25^(hi) and CD25^(int) sorting.

FIGS. 10A-10E demonstrate that TNFRSF25 agonists protect might fromdextran-sodium sulfate induced colitis, a mouse model of Crohn'sdisease. C57BL/6 mice or TL1A knockout mice were provided with 3%dextran sodium sulfate (DSS) dissolved in drinking water ad libitum for7 days. In some experiments, mice were treated on experimental day 0with IgG isotype control antibody or with the TNFRSF25 agonisticantibody, clone 4C12. Weight was monitored daily beginning 4 days priorto provision of DSS (experimental day-4). On day-4, one group of micewas treated with the TNFRSF25 agonistic antibody, clone 4C12, byintraperitoneal injection (20 μg/mouse), with the other treated withhamster IgG isotype control antibody. Mortality was measured whenanimals lost ≧20% of starting body weight (FIG. 10A). In someexperiments, animals were sacrifice at experimental day 5, and total RNAwas prepared using the RNeasy miniprep kit (Qiagen) from flash-frozen,PBS-washed, colonic tissue. RNA was subsequently reverse transcribed(Quantitect RT, Qiagen) and cDNA was amplified by real-time PCR usingTaqman (Applied Biosystems) probes for the indicated transcripts (FIG.10B). Data are shown as the fold change in expression in TL1A knockoutmice as compared to C57BL/6 control mice. The percentage body weightloss was monitored and plotted over the course of the study in eachexperimental group (FIG. 10C). In experiments where animals weresacrificed on experimental day 5 for RNA isolation, mesenteric lymphnodes were isolated for analysis by flow cytometry for the proportion ofCD4⁺ cells expressing the transcription factor FoxP3, indicative of theregulatory T cell pool (FIG. 10D). Finally, reverse transcription wasperformed using RNA isolated from the indicated treatment groups asdescribed for FIG. 10B and subjected to RT-PCR for the indicatedtranscripts. Error bars indicate mean±S.E.M. for ≧3 mice pet experimentand a minimum of 2 experiments per panel.

FIG. 11 demonstrates that TNFRSF25 agonists delay acute rejection ofallogeneic hearts in a heterotopic heart transplant model in mice. Tostudy tolerance induction by 4C12 expanded natural Treg a heterotopicheart transplant model was used which is well described for tolerancestudies. Hearts from CBA/J mice (H2^(d)) were transplanted into theabdomen of C57BL/6 mice (H2^(b)) on day 0. On day-4, one group of micewas treated with the TNFRSF25 agonistic antibody, clone 4C12, byintraperitoneal injection (20 μg/mouse), with the other treated withhamster IgG isotype control antibody. At the time of transplant Tregexpansion in the blood was confirmed in the 4C12 treated group.Allograft survival was monitored by palpating the heart manually and thepulse was graded on a scale from 0 to 4 (0=no pulse; 1=very mild;2=mild; 3=moderate; 4=strong). Rejection is defined as cessation ofpalpable heart beat. At the time of rejection (=when the heartbeatstopped) the graft was removed, formalin fixed and submitted forpathologic examination. Loss of graft function within 48 h of transplantis considered a technical failure (<5%) and omitted from furtheranalysis.

FIG. 12 demonstrates that TNFRSF25 agonists selectively expand natural;but not induced, T regulatory cells. CD4⁺FoxP3⁻ T cells were isolatedfrom mice expressing a FoxP3-RFP reporter gene and cultured in vitro for5 days in the presence of IL-2, TGF-beta, anti-CD3 antibody and retinoicacid according to standard protocols. At the conclusion of the cultureperiod the viable cells contained within the CD4⁺ population contained70-85% CD4⁺FoxP3-RFP⁺ induced regulatory T cells (iTreg). These cellswere purified by high-speed cell sorting. Concurrently, total CD4⁺ cellswere purified from mice expressing a FoxP3-GFP reporter gene (thesecells therefore contain a mixture of iTreg and so-called, thymicallyderived, natural regulatory T cells (nTreg)). iTreg (6×10⁵ iTreg cellswere mixed with total CD4⁺ cells isolated from FoxP3-GFP mice containing8×10⁵ Treg) and adoptively transferred (by intravenous injection) intoCD4^(−/−) recipient mice. After 2 days, CD4^(−/−) recipient micecontaining a mixture of RFP-positive iTreg and GFP-positive nTreg weretreated with either 4C12 or IgG isotype control antibodies (20 μg/mouse,by intraperitoneal injection). Five days later, the proportion ofRFP-positive iTreg and GFP-positive total Treg (containing the onlysource of nTreg) was determined by flow cytometry of isolatedsplenocytes (FIG. 12).

DETAILED DESCRIPTION

The present invention is described with reference to the attachedfigures, wherein like reference numerals are used throughout the figuresto designate similar or equivalent elements. The figures are not drawnto scale and they are provided merely to illustrate the instantinvention. Several aspects of the invention are described below withreference to example applications for illustration. It should beunderstood that numerous specific details, relationships, and methodsare set forth to provide a full understanding of the invention. Onehaving ordinary skill in the relevant art, however, will readilyrecognize that the invention can be practiced without one or more of thespecific details or with other methods. The present invention is notlimited by the illustrated ordering of acts or events, as some acts mayoccur in different orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the moleculesdisclosed herein e.g. 4C2 is not limited to mice but the human antibodyis preferred, which in some embodiments relate to mammalian nucleic acidand amino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inpreferred embodiments, the genes or nucleic acid sequences are human.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample; “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

A “T regulatory cell” or “Treg cell” or “Tr cell” refers to a cell thatcan modulate a T cell response. Treg cells express the transcriptionfactor Foxp3, which is not upregulated upon T cell activation anddiscriminates Tregs from activated effector cells. Tregs are identifiedby the cell surface markers CD25, CTLA4, and GITR. Several Treg subsetshave been identified that have the ability to inhibit autoimmune andchronic inflammatory responses and to maintain immune tolerance intumor-bearing hosts. These subsets include interleukin 10-(IL-10-)secreting T regulatory type 1 (Tr1) cells, transforming growthfactor-β-(TGF-β-) secreting T helper type 3 (Th3) cells, and “natural”CD4⁺/CD25⁺ Tregs (Trn) (Fehervari and Sakaguchi. J. Clin. Invest. 2004,114:1209-1217; Chen et al. Science. 1994, 265: 1237-1240; Groux et al.Nature. 1997, 389: 737-742).

“TNFR25 agonist”, “TNFR25 agent”, “TNFR25 composition” are usedinterchangeably herein and refer to a substance that binds to the TNFR25receptor and triggers a response in the cell on which the TNFR25receptor is expressed similar to a response that would be observed byexposing the cell to a natural TNFR25 ligand, e.g., TL1A. An agonist isthe opposite of an antagonist in the sense that while an antagonist mayalso bind to the receptor, it fails to activate the receptor andactually completely or partially blocks it from activation by endogenousor exogenous agonists. A partial agonist activates a receptor but doesnot cause as much of a physiological change as does a full agonist.Alternatively, another example of a TNFR25 agonist is an antibody thatis capable of binding and activating TNFR25. An example of an anti-TNFRantibody is 4C12 (agonist). (Deposited under the Budapest Treaty onBehalf of: University of Miami; Date of Receipt of seeds/strain(s) bythe ATCC®: May 5, 2009; ATCC®Patent Deposit Designation: PTA-10000.Identification Reference by Depositor: Hybridoma cell line; 4C12; Thedeposit was tested Jun. 4, 2009 and on that date, the seeds/strain(s)were viable. International Depository Authority: American Type CultureCollection (ATCC®), Manassas, Va., USA).

“TNFR25 antagonist” is referred to herein as a substance that inhibitsthe normal physiological function of a TNFR25 receptor. Such agents workby interfering in the binding of endogenous receptor agonists/ligandssuch as TL1A, with TNFR25 receptor.

TNFR25 antagonists or agonists may be in the form of aptamers.“Aptamers” are DNA or RNA molecules that have been selected from randompools based on their ability to bind other molecules. The aptamer bindsspecifically to a target molecule wherein the nucleic acid molecule hassequence that comprises a sequence recognized by the target molecule inits natural setting. Alternately, an aptamer can be a nucleic acidmolecule that binds to a target molecule wherein the target moleculedoes not naturally bind to a nucleic acid. The target molecule can beany molecule of interest. For example, the aptamer can be used to bindto a ligand-binding domain of a protein, thereby preventing interactionof the naturally occurring ligand with the protein. This is anon-limiting example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brodyand Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100,2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science287:820, 2000; and Jayasena, Clinical Chem. 45:1628, 1999).

As used herein, the term “antibody” is inclusive of all species,including human and humanized antibodies and the antigenic target, forexample, TNFR25, can be from any species. Thus, an antibody, forexample, anti-TNFR25 can be mouse anti-human TNFR25, goat anti-humanTNFR25; goat anti-mouse TNFR25; rat anti-human TNFR25; mouse anti-ratTNFR25 and the like. The combinations of antibody generated in a certainspecies against an antigen target, e.g. TNFR25, from another species, orin some instances the same species (for example, in autoimmune orinflammatory response) are limitless and all species are embodied inthis invention. The term antibody is used in the broadest sense andincludes fully assembled antibodies, monoclonal antibodies (includinghuman, humanized or chimeric antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)₂, Fv, single chainantibodies, diabodies), comprising complementarity determining regions(CDRs) of the foregoing as long as they exhibit the desired biologicalactivity.

“Target molecule” includes any macromolecule, including protein,carbohydrate, enzyme, polysaccharide, glycoprotein, receptor, antigen,antibody, growth factor; or it may be any small organic moleculeincluding a hormone, substrate, metabolite, cofactor, inhibitor, drug,dye, nutrient, pesticide, peptide; or it may be an inorganic moleculeincluding a metal, metal ion, metal oxide, and metal complex; it mayalso be an entire organism including a bacterium, virus, and single-celleukaryote such as a protozoon.

“Treating” or “treatment” of a state, disorder or condition includes:(1) Preventing or delaying the appearance of clinical or sub-clinicalsymptoms of the state, disorder or condition developing in a mammal thatmay be afflicted with or predisposed to the state, disorder or conditionbut does not yet experience or display clinical or subclinical symptomsof the state, disorder or condition; or (2) Inhibiting the state,disorder or condition, i.e., arresting, reducing or delaying thedevelopment of the disease or a relapse thereof (in case of maintenancetreatment) or at least one clinical or sub-clinical symptom thereof; or(3) Relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms. The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

“Patient” or “subject” refers to mammals and includes human andveterinary subjects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Immune Response Modulation

Characteristics of CD4 T cell subsets: CD4 T cells upon activation andexpansion develop into different T helper (T_(H)) cell subsets withdifferent cytokine profiles and distinct effector functions. Appropriatedifferentiation of T_(H) cells into effector subsets best suited forhost defense against an invading pathogen is of critical importance tothe immune system. CD4 T cells differentiate into at least four knownsubsets, three effector subsets (T_(H)1, T_(H)2 and T_(H)17) and one Tregulatory subset (Treg). Based on the cytokines that they produce, Tcells were historically divided into T_(H)1 and T_(H)2 cells, and thishas provided a framework to understand how specific cytokine milieusproduced by cells of the innate immune system guide the development ofadaptive immunity. T_(H)1 cells, which are potently induced by dendriticcells (DC) secreting IL-12, are characterized by the expression of thelineage-specific transcription factor T-bet (T box 21) and theproduction of IFN-γ. T_(H)2 cells, which depend on IL-4 duringdifferentiation and lack of IL-12, produce IL-4, IL-5, IL-9, and IL-13and are characterized by the expression of the transcription factorGATA-3. Importantly, in the past five years, a third subset ofIL-17-producing effector T helper cells, called T_(H)17 cells, has beendiscovered and characterized and is specified by expression of thetranscription factor RORγt.

T_(H)17 cells produce IL-17, IL-17F, and IL-22. By secreting theseeffector cytokines, T_(H)17 cells induce a massive tissue reaction dueto the broad distribution of the IL-17 and IL-22 receptors. T_(H)17cells also secrete IL-21 to communicate with the cells of the immunesystem. Synergy between the cytokines transforming growth factor betaisoform 1 (TGF-β) and interleukin (IL)-6 induces development of T_(H)17cells in mice and humans, while IL-23 supports expansion of these cells.The differentiation factors (TGF-3 plus IL-6 or IL-21), the growth andstabilization factor (IL-23), and the transcription factors (STAT3,ROR-γt (ROR-c), and ROR-a) involved in the development of T_(H)17 cellshave only recently been identified. The participation of TGF-β in thedifferentiation of T_(H)17 cells places the T_(H)17 lineage in closerelationship with CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_(reg)) sinceTGF-β also induces differentiation of naive T cells into Foxp3⁺ Treg inthe peripheral immune compartment. Treg cells are a specializedsubpopulation of T cells that act to suppress activation of the immunesystem and thereby maintain immune system homeostasis and tolerance toself-antigens. Development of Treg cells, which are capable ofsuppressing autoimmune disease, is reciprocally related to T_(H)17cells, which can drive immune responses, including autoimmune responses.Treg cells can be identified by their unique expression of thetranscription factor forkhead box P3 (Foxp3). Importantly, so far as isknown, there are two phenotypically identical populations of CD4⁺CD25⁺Treg—natural and adaptive. Natural CD4⁺CD25⁺ Treg cells arise in thethymus under homeostatic conditions to safeguard against autoimmunity.Adaptive CD4⁺CD25⁺ Treg cells arise during inflammatory processes suchas infections and cancers and suppress immunity through heterogeneousmechanisms that include direct contact or the production of solublefactors such as IL-10 and TGF-β.

Tumor Necrosis Factor Receptor 25 (TNFR25):

Interchangeably referred to herein as Death receptor 3 (DR3), is aregulator of T cell function. Death receptor 3 (DR3) (Chinnaiyan et al.,Science 274:990, 1996) is a member of the TNF-receptor family. It isalso known as TRAMP (Bodmer et al., Immunity 6:79, 1997), wsl-1 (Kitsonet al., Nature 384:372, 1996), Apo-3 (Marsters et al., Curr Biol 6:1669,1996), and LARD (Screaton et al., Proc Nall Acad Sci USA 94:4615, 1997)and contains a typical death domain. Transfection of 293 cells withhuman DR3 (hDR3) induced apoptosis and activated NF-κB. Multiple splicedforms of human DR3 mRNA have been observed, indicating regulation at thepost transcriptional level (Screaton et al., Proc Natl Acad Sci USA94:4615, 1997).

CD4⁺FoxP3⁺ regulatory T cells (Treg) can suppress the activity ofautoreactive effector T cells that escape negative selection in thethymus. Tregs are sufficient to prevent or delay autoimmune pathology inexperimental models of IBD, asthma and EAE. TNFR25 signaling blocksTregs from inhibiting CD4⁺CD25⁻ but not antigen-specific CD8⁺ cells invitro. Interestingly, transgenic mice expressing full-length TNFR25under the CD2 promoter express high levels of T_(H)2 and T_(H)17cytokines and have decreased cellularity in secondary lymphoid tissues.The coincidence of decreased regulatory T cell activity, decreasedcellularity and increased cytokine production in TNFR25 transgenic micesuggests that TNFR25 signaling may be both pro- and anti-inflammatorydepending on the context in which TNFR25 signals are received.

Briefly, the experiments conducted herein, showed the unexpected findingthat in vivo stimulation of TNFR25 leads to the rapid and systemicexpansion of the CD4⁺FoxP3⁺ regulatory T cell pool. Antibody (4C12)induced Treg expansion occurred independently of exogenous antigen andresulted in a 3-4 fold increase in the percentage of Tregs out of totalCD4⁺ cells within 4 days of administration. This Treg expansion resultedpredominantly from the proliferation of CD4⁺FoxP3⁺CD25^(HU) cells, isdurable, and does not contract to unstimulated levels for two weeks.4C12 expanded Tregs retain TGF mediated effector T cell suppressorfunctions ex vivo; however continued TNFR25-signaling abrogates thesuppressive activity of 4C12 expanded Tregs. Without wishing to be boundby theory, these findings indicate that TNFR25 signaling in Tregs hasthe dual function of increasing Treg proliferation and inhibition ofTreg suppressor activity. Inhibition of Treg suppression by TNFR25signaling is highly plastic and can be restored or maintained followingremoval or continuation of TNFR25 signaling in Tregs, respectively. Theaddition of this information to the role of TNFR25 as a costimulator ofT_(H)2 and T_(H)17 responses indicates that the role of TNFR25 in immunesignaling is to simultaneously enhance effector cell function during theinduction of an inflammatory response and accelerate resolution ofinflammation via a regionally expanded pool of Tregs that regainsuppressive activity following removal of the inflammatory stimulus.Taken together, these findings evidence that TNFR25 targeted therapiesmay be valuable to either enhance or inhibit immune activation,depending on the inflammatory context in which they are administered;with broad implications to the fields of autoimmune disease, chronicinfection, transplantation and cancer.

In one preferred embodiment, a method of regulating an immune responsein vivo comprises administering to a patient in need thereof, at leastone agent which modulates tumor necrosis factor superfamily receptor 25(TNFRSF25; TNFR25; DR3) function. The preferred function is TNFRSF25mediated signaling which results in the induction of regulatory T cell(Treg) proliferation. Stimulation of the TNFRSF25 molecule induces Tregcells which suppress an immune response. However, continued stimulationof the TNFRSF25 molecule abrogates the suppressive activity of the Tregcells, thus, regulating an immune response.

Signaling through the TNF-receptor superfamily member 25 (TNFRSF25, DR3)on CD4⁺ T cells, where it is constitutively expressed, enhances T₁₁2 andT₁₁17 cytokine production and contributes to pathological inflammationin disease models of asthma, inflammatory bowel disease, multiplesclerosis (MS), and rheumatoid arthritis.

In preferred embodiments regulating the immune response in vivo treatsdiseases or disorders associated with an immune response. Such diseasesor disorders comprise, for example: rejection reactions bytransplantation of organs or tissues such as the heart, kidney, liver,bone marrow, skin, cornea, lung, pancreas, small intestine, limb,muscle, nerve, intervertebral disc, trachea, myoblast, cartilage, etc.;graft-versus-host reactions following bone marrow transplantation;autoimmune diseases such as rheumatoid arthritis, systemic lupuserythematosus, Hashimoto's thyroiditis, multiple sclerosis, myastheniagravis, type I diabetes, etc.; infections caused by pathogenicmicroorganisms (e.g. Aspergillus fumigatus, Fusarium oxysporum,Trichophyton asteroides, etc.); inflammatory or hyperproliferative skindiseases or cutaneous manifestations of immunologically mediateddiseases (e.g. psoriasis, atopic dermatitis, contact dermatitis,eczematoid dermatitis, seborrheic dermatitis, lichen planus, pemphigus,bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema,vasculitides, erythema, dermal eosinophilia, lupus erythematosus, acne,and alopecia areata); autoimmune diseases of the eye (e.g.keratoconjunctivitis, vernal conjunctivitis, uveitis associated withBehcet's disease, keratitis, herpetic keratitis, conical keratitis,corneal epithelial dystrophy, keratoleukoma, ocular premphigus, Mooren'sulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome,keratoconjunctivitis sicca (dry eye), phlyctenule, iridocyclitis,sarcoidosis, endocrine ophthalmopathy, etc.); reversible obstructiveairways diseases [asthma (e.g. bronchial asthma, allergic asthma,intrinsic asthma, extrinsic asthma, and dust asthma), particularlychronic or inveterate asthma (e.g. late asthma and airwayhyper-responsiveness) bronchitis, etc.; mucosal or vascularinflammations (e.g. gastric ulcer, ischemic or thrombotic vascularinjury, ischemic bowel diseases, enteritis, necrotizing enterocolitis,intestinal damages associated with thermal burns, leukotrieneB4-mediated diseases); intestinal inflammations/allergies (e.g. coeliacdiseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn'sdisease and ulcerative colitis); food-related allergic diseases withsymptomatic manifestation remote from the gastrointestinal tract (e.g.migrain, rhinitis and eczema); renal diseases (e.g. intestitialnephritis, Goodpasture's syndrome, hemolytic uremic syndrome, anddiabetic nephropathy); nervous diseases (e.g. multiple myositis,Guillain-Barre syndrome, Meniere's disease, multiple neuritis, solitaryneuritis, cerebral infarction, Alzheimer's diseases Parkinson'sdiseases, amyotrophic lateral sclerosis (ALS) and radiculopathy);cerebral ischemic disease (e.g., head injury, hemorrhage in brain (e.g.,subarachnoid hemorrhage, intracerebral hemorrhage), cerebral thrombosis,cerebral embolism, cardiac arrest, stroke, transient ischemic attack(TIA), hypertensive encephalopathy, cerebral infarction); endocrinediseases (e.g. hyperthyroidism, and Basedow's disease); hematic diseases(e.g. pure red cell aplasia, aplastic anemia, hypoplastic anemia,idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia,agranulocytosis, pernicious anemia, megaloblastic anemia, andanerythroplasia); bone diseases (e.g. osteoporosis); respiratorydiseases (e.g. sarcoidosis, pulmonary fibrosis, and idiopathicinterstitial pneumonia); skin diseases (e.g. dermatomyositis, leukodermavulgaris, ichthyosis vulgaris, photosensitivity, and cutaneous T-celllymphoma); circulatory diseases (e.g. arteriosclerosis, atherosclerosis,aortitis syndrome, polyarteritis nodosa, and myocardosis); collagendiseases (e.g. scleroderma, Wegener's granuloma, and Sjogren'ssyndrome); adiposis; eosinophilic fasciitis; periodontal diseases (e.g.damage to gingiva, periodontium, alveolar bone or substantia osseadentis); nephrotic syndrome (e.g. glomerulonephritis); male patternalopecia, alopecia senile; muscular dystrophy; pyoderma and Sezarysyndrome; chromosome abnormality-associated diseases (e.g. Down'ssyndrome); Addison's disease; active oxygen-mediated diseases [e.g.organ injury (e.g. ischemic circulation disorders of organs (e.g. heart,liver, kidney, digestive tract, etc.) associated with preservation,transplantation, or ischemic diseases (e.g. thrombosis, cardialinfarction, etc.)); intestinal diseases (e.g. endotoxin shock,pseudomembranous colitis, and drug- or radiation-induced colitis); renaldiseases (e.g. ischemic acute renal insufficiency, chronic renalfailure); pulmonary diseases (e.g. toxicosis caused by pulmonary oxygenor drugs (e.g. paracort, bleomycin, etc.), lung cancer, and pulmonaryemphysema); ocular diseases (e.g. cataracta, iron-storage disease(siderosis bulbi), retinitis, pigmentosa, senile plaques, vitreousscarring, corneal alkali burn); dermatitis (e.g. erythema multiforme,linear immunoglobulin A bullous dermatitis, cement dermatitis); andother diseases (e.g. gingivitis, periodontitis, sepsis, pancreatitis,and diseases caused by environmental pollution (e.g. air pollution),aging, carcinogen, metastasis of carcinoma, and hypobaropathy)];diseases caused by histamine release or leukotriene C4 release;restenosis of coronary artery following angioplasty and prevention ofpostsurgical adhesions; autoimmune diseases and inflammatory conditions(e.g., primary mucosal edema, autoimmune atrophic gastritis, prematuremenopause, male sterility, juvenile diabetes mellitus, pemphigusvulgaris, pemphigoid, sympathetic ophthalmitis, lens-induced uveitis,idiopathic leukopenia, active chronic hepatitis, idiopathic cirrhosis,discoid lupus erythematosus, autoimmune orchitis, arthritis (e.g.arthritis deformans), or polychondritis); Human Immunodeficiency Virus(HIV) infection, AIDS; allergic conjunctivitis; hypertrophic cicatrixand keloid due to trauma, burn, or surgery.

In one preferred embodiment, the agent is administered in a single doseor spread out over a period of time in order to maintain the suppressiveeffects of Treg cells. For example, treatment of autoimmune diseases.

In another preferred embodiment, the agent is administered in multipleor a plurality of doses so that the suppressive effects of the Tregcells are abrogated. For example, in the treatment of cancer, viraldiseases or other diseases requiring an immune mediated clearing ofabnormal cells.

In another preferred embodiment, the agent is administered in anextended release formulation so as to provide a constant stimulation ofTNFRSF25 so as to abrogate the suppression of the immune system by Tregcells.

In another preferred embodiment, at least one agent stimulates signalingof TNFRSF25 and induces proliferation of CD4⁺FoxP3⁺CD25^(int) cells in apatient. The suppressive effects of the Treg cells can be monitored andthe doses of the agent can be adjusted to maintain the suppressiveeffects in diseases or conditions wherein a decreased immune response isdesired, for example, autoimmunity, transplantation rejection and thelike.

In another preferred embodiment, at least one agent stimulates signalingof TNFRSF25 and induces proliferation of thymically induced, but notperipherally induced, Treg cells. The suppressive effects of the Tregcells can be monitored and the doses of the agent can be adjusted tomaintain the suppressive effects in diseases or conditions wherein adecreased immune response is desired, for example, autoimmunity,transplantation rejection and the like.

In another preferred embodiment, at least one agent stimulates thesignaling of TNFRSF25 and induces the proliferation of both thymicallyand peripherally induced Treg cells, where the cognate antigenrecognized by the peripherally induced Tregs is known to be present. Thesuppressive effects of the Treg cells can be monitored and the doses ofthe agent can be adjusted to maintain the suppressive effects indiseases or conditions wherein a decreased immune response is desired,for example, autoimmunity, transplantation rejection and the like.

In another preferred embodiment, one or a combination of agents can beadministered to a patient to modulate their immune response. Forexample, a patient may receive one or more agents in a therapeuticallyeffective dose as determined by the proliferation of CD4⁺FoxP3⁺ cells ina patient, or any other assay that measures the desired response. Forexample immunoassays, biomarker detection, FACS, immuno blots,hybridization, PCR etc. The Treg cells can be identified as, forexample, CD4⁺FoxP3⁺ cells. In some aspects there is co-expression ofCD103. Expression of CD103 by Tregs contributes to the retention oftissues within tissues. Agents known not to interfere with TNFRSF25mediated Treg proliferation include rapamycin.

In another preferred embodiment, the agent modulating TNFRSF25 signalingcomprises at least one of: an antibody, an aptamer, a ligand, smallmolecule, peptide, protein, oligonucleotide, polynucleotide, organic orinorganic molecule.

In one preferred embodiment, the agent is an agonist of TNFRSF25.

In another preferred embodiment, a method of suppressing an immuneresponse in vivo comprises administering to a patient in need thereof,at least one agent which modulates tumor necrosis factor superfamilyreceptor 25 (TNFRSF25; TNFR25; DR3) mediated signaling function; and,inducing a suppressive regulatory T cell (Treg) expansion.

In another preferred embodiment, the agent modulates tumor necrosisfactor superfamily receptor 25 signaling and inhibits the suppressiveactivity of CD4⁺FoxP3⁺ regulatory T cells.

In another preferred embodiment, a composition for modulating an immuneresponse comprising an agent which modulates TNFRSF25 signaling. In oneembodiment, the agent is administered to a patient so that suppressorTregs suppress an immune response. In another preferred embodiment, theagent is administered to a patient in a dose or under conditions whichabrogate the signal leading to an inhibition of suppressor Treg cells sothat an immune response is mounted.

In another preferred embodiment, a method of treating cancer in vivocomprising administering to a patient in need thereof, at least oneagent which modulates tumor necrosis factor superfamily receptor 25(TNFRSF25; TNFR25; DR3) function signaling at doses and conditions whichprovide continual stimulation of the TNFRSF25 which abrogates thesuppressive effects of the Treg cells an immune response to cancer canbe induced.

In another preferred embodiment, modulation of immune cells andsubsequent responses comprises a method of treating a patient with adisease such as for example, cancer, viral disease, or disease caused byany infectious organism wherein an anti-TNFR25 composition, isadministered to a patient, and modulates the functions of the immunecells, for example, proliferation of a lymphocyte wherein thatlymphocyte had been previously suppressed or attenuated, or in caseswhere the immune response is normal but the enhancement of theenhancement of the immune response results in more effective and fastertreatment of a patient. Negative regulatory pathway, and not lack ofinherent tumor immunogenicity (i.e., the ability of the unmanipulatedtumors to stimulate protective immunity), play an important role inpreventing the immune-mediated control of tumor progression. Thetherapeutic implication is that counteringimmune-attenuating/suppressive regulatory circuits contributes tosuccessful immune control of cancer and is as, if not more, importantthan developing potent vaccination protocols.

Tumor Vaccines:

As such, TNFR25 agonists are effective biological response modifiers, infor example, for tumor vaccines because they boost T cell activation andthe cellular immune response to a tumor specific antigen, whereas TNFR25antagonists block or inhibit T cell activation. Therefore, anotheraspect of the invention relates to methods and therapeutic agents thatincrease the effectiveness of a tumor vaccine.

Tumor vaccines attempt to the use of elements of the body's naturalimmune system to fight cancer. Tumor vaccines contain one or more tumorspecific antigens and may contain an adjuvant and biological responsemodifiers. A tumor specific antigen is a polypeptide that issubstantially limited to expression in or on tumor cells and which canbe used to stimulate an immune response intended to target those tumorcells. Different types of vaccines are used to treat different types ofcancer. For an antigenic composition to be useful as a vaccine, anantigenic composition must induce an immune response to the antigen in acell or tissue. As used herein, an “antigenic composition” may comprisean antigen (e.g., a peptide or polypeptide), a nucleic acid encoding anantigen (e.g., an antigen expression vector), or a cell expressing orpresenting an antigen.

The enhancement of the immune response to a vaccine or other antigenicstimulant can be measured by any conventional method, such as forexample, proliferation assays, cytokine secretion, types of cytokinessecreted, cytotoxic T lymphocyte assays, ELISAs, RIA and the like. Theenhanced immune response can also be detected by monitoring thetreatment. For example, in the case of treating cancer, an enhancedimmune response could also be monitored by observing one or more of thefollowing effects: (1) inhibition, to some extent, of tumor growth,including, (i) slowing down (ii) inhibiting angiogenesis and (ii)complete growth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion and/or (8) relief, to some extent, of the severity or number ofone or more symptoms associated with the disorder.

In another preferred embodiment, the anti-TNFR25 can be administered asa vector construct expressing anti-TNFR25 antibodies. In addition, thevector construct can contain nucleotide sequences encoding cytokines,such as granulocyte macrophage colony stimulating factor (GM-CSF),interleukin-12 (IL-12) and co-stimulatory molecules such B7-1, B7-2,CD40. The cytokines can be used in various combinations to fine-tune theresponse of the subject's immune system, including both antibody andcytotoxic T lymphocyte responses, to bring out the specific level ofresponse needed to control or eliminate the infection or disease state.The polynucleotide can also encode a fusion product containing anantigenic polypeptide, for example, an anti-tumor antigen, anti-viralantigen and the like, and a co-stimulatory molecule, such as CTLA-4.Examples of suitable vectors comprise viral vectors which include poliovirus, pox viruses such as vaccinia, canary pox, and fowl pox, herpesviruses, including catfish herpes virus, adenovirus-associated vector,and retroviruses. Exemplary bacterial vectors include attenuated formsof Salmonella, Shigella, Edwardsiella ictaluri, Yersinia ruckerii, andListeria monocytogenes. L. monocytogenes may also be valuable as aresearch tool used to stimulate the expansion of Tregs in animals sothat they can be harvested at a later time point with an increased yieldof Tregs per animal.

Combination Therapies

In a preferred embodiment, the enhancement or up-regulation of an immuneresponse can be combined with one or more therapies. The anti-TNFR25antibody, for example, can be administered prior to, concurrently with,or after a course of treatment with one or more agents or methods oftreatment.

In another embodiment, the TNFR25 compositions can be administered toautologous cells, allow the cells to expand and then re-infuse the cellsinto the patient.

The TNFR25 stimulating agents can be administered in a pharmaceuticalcomposition, as a polynucleotide in a vector, liposomes, nucleic acidspeptides and the like.

In another preferred embodiment, the TNFR25 stimulating agents can beadministered with one or more or additional pharmacologically activeagents. As used herein, the term “pharmacologically active agent” refersto any agent, such as a drug, capable of having a physiologic effect(e.g., a therapeutic or prophylactic effect) on prokaryotic oreukaryotic cells, in vivo or in vitro, including, but withoutlimitation, chemotherapeutics, toxins, radiotherapeutics,radiosensitizing agents, gene therapy vectors, antisense nucleic acidconstructs or small interfering RNA, imaging agents, diagnostic agents,agents known to interact with an intracellular protein, polypeptides,and polynucleotides.

The additional pharmacologically active agent can be selected from avariety of known classes of drugs, including, for example, analgesics,anesthetics, anti-inflammatory agents, anthelmintics, anti-arrhythmicagents, antiasthma agents, antibiotics (including penicillins),anticancer agents (including Taxol), anticoagulants, antidepressants,antidiabetic agents, antiepileptics, antihistamines, antitussives,antihypertensive agents, antimuscarinic agents, antimycobacterialagents, antineoplastic agents, antioxidant agents, antipyretics,immunosuppressants, immunostimulants, antithyroid agents, antiviralagents, anxiolytic sedatives (hypnotics and neuroleptics), astringents,bacteriostatic agents, beta-adrenoceptor blocking agents, blood productsand substitutes, bronchodilators, buffering agents, cardiac inotropicagents, chemotherapeutics, contrast media, corticosteroids, coughsuppressants (expectorants and mucolytics), diagnostic agents,diagnostic imaging agents, diuretics, dopaminergics (antiparkinsonianagents), free radical scavenging agents, growth factors, haemostatics,immunological agents, lipid regulating agents, muscle relaxants,proteins, peptides and polypeptides, parasympathomimetics, parathyroidcalcitonin and biphosphonates, prostaglandins, radio-pharmaceuticals,hormones, sex hormones (including steroids), time release binders,anti-allergic agents, stimulants and anoretics, steroids,sympathomimetics, thyroid agents, vaccines, vasodilators, and xanthines.

The additional pharmacologically active agent need not be a therapeuticagent. For example, the agent may be cytotoxic to the local cells towhich it is delivered but have an overall beneficial effect on thesubject. Further, the agent may be a diagnostic agent with no directtherapeutic activity per se, such as a contrast agent for bioimaging.

Chemotherapy:

The TNFR25 compositions can be administered with chemotherapy.Administration of for example, anti-TNFR25 would likely result in thedecreased need of chemotherapy, or if chemotherapy is still required orrecommended, the doses would be lower, thereby alleviating some of theadverse side effects of these chemotherapeutic agents. Cancer therapiesalso include a variety of combination therapies with both chemical andradiation based treatments. Combination chemotherapies include, forexample, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate, or any analogor derivative variant of the foregoing.

Radiotherapy:

The compositions can be combined with radiotherapy. Other factors thatcause DNA damage and have been used extensively include what arecommonly known as .gamma.-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. Other forms of DNA damaging factors arealso contemplated such as microwaves, proton beam irradiation (U.S. Pat.No. 5,760,395 and U.S. Pat. No. 4,870,287) and UV-irradiation. It ismost likely that all of these factors effect a broad range of damage onDNA, on the precursors of DNA, on the replication and repair of DNA, andon the assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

Immunotherapy:

The anti-TNFR25 agents can be combined with other forms ofimmunotherapy. For example, in the context of cancer treatment,immunotherapeutics, generally, rely on the use of immune effector cellsand molecules (e.g., monoclonal antibodies) to target and destroy cancercells. Trastuzumab (HERCEPTIN™) or bevacizumab (AVASTIN™) is such anexample. The immune effector may be, for example, an antibody specificfor some marker on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually effect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells. The combination of therapeutic modalities, i.e., direct cytotoxicactivity and enhancement of tat immune effector response by for example,anti-TNFR25 antibody, would provide therapeutic benefit in the treatmentof cancer.

The antigen specific immune response would target one or more tumorantigens and the administration of the TNFR25 compositions would enhancethe immune response directed to these tumor antigens. Many tumor markersexist and any of these may be suitable for targeting in the context ofthe present invention. Common tumor markers include carcinoembryonicantigen, prostate specific antigen, urinary tumor associated antigen,fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl LewisAntigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb Band p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growthfactors such as FLT3 ligand. Combining immune stimulating molecules,either as proteins or using gene delivery in combination with a tumorsuppressor such as MDA-7 enhance anti-tumor effects.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow. Preferably, human monoclonal antibodies areemployed in passive immunotherapy, as they produce few or no sideeffects in the patient.

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogeneic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant. In melanomaimmunotherapy, those patients who elicit high IgM response often survivebetter than those who elicit no or low IgM antibodies. IgM antibodiesare often transient antibodies and the exception to the rule appears tobe anti-ganglioside or anti-carbohydrate antibodies.

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated by,lymphokines such as IL-2 or transduced with genes for tumor necrosis.The TNFR25 compositions, for example anti-TNFR25 antibody, areadministered or cultured with the cells which are then re-infused. Toachieve this, one would administer to an animal, or human patient, animmunologically effective amount of activated lymphocytes in combinationwith anti-TNFR25 and, optionally, with an adjuvant-incorporatedantigenic peptide composition. The activated lymphocytes will mostpreferably be the patient's own cells that were earlier isolated from ablood or tumor sample and activated (or “expanded”) in vitro. This formof immunotherapy has produced several cases of regression of melanomaand renal carcinoma, but the percentage of responders were few comparedto those who did not respond. The anti-TNFR25 can be administered to apatient, after re-infusion to the cells under a regimen that can bedetermined by the treating physician or nurse practitioner.

Immunosuppressants:

The administration of one or more TNFRSF25 agents can be administeredwith one or more immunosuppressants where it is desired to maintain asuppressed immune response (e.g. autoimmune diseases). Examples ofimmunosuppressants, include without limitation: mycophenolic acid,azathioprine, cyclosporine A, FK506, FK520, Elidel; tacrolimus andsiroliinus; minocycline; leflunomide; or methotrexate.

Surgery:

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well

Other Agents:

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents that affect theupregulation of cell surface receptors and GAP junctions, cytostatic anddifferentiation agents, the inhibition of cell adhesion, and theincrease in sensitivity of the hyperproliferative cells to apoptoticinducers or other agents. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL (Apo-2 ligand) would potentiate the enhancing abilities of thepresent invention. Increases in intercellular signaling by elevating thenumber of GAP junctions would increase the proliferative effects ondesired cell populations.

Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosisfactor (TNF) cytokine family. TRAIL activates rapid apoptosis in manytypes of cancer cells, yet is not toxic to normal cells. TRAIL mRNAoccurs in a wide variety of tissues. Most normal cells appear to beresistant to TRAIL's cytotoxic action, suggesting the existence ofmechanisms that can protect against apoptosis induction by TRAIL. Thefirst receptor described for TRAIL, called death receptor 4 (DR4),contains a cytoplasmic “death domain”; DR4 transmits the apoptosissignal carried by TRAIL. Additional receptors have been identified thatbind to TRAIL. One receptor, called DR5, contains a cytoplasmic deathdomain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs areexpressed in many normal tissues and tumor cell lines. Decoy receptorssuch as DcR1 and DcR2 have been identified that prevent TRAIL frominducing apoptosis through DR4 and DR5. These decoy receptors thusrepresent a novel mechanism for regulating sensitivity to apro-apoptotic cytokine directly at the cell's surface. The preferentialexpression of these inhibitory receptors in normal tissues suggests thatTRAIL may be useful as an anticancer agent that induces apoptosis incancer cells while sparing normal cells.

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, an enhancement of theimmune response provides an alternative approach.

Another form of therapy includes hyperthermia, which is a procedure inwhich a patient's tissue is exposed to high temperatures (up to 106°F.). External or internal heating devices may be involved in theapplication of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radio frequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets:Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

Administration of Compositions

The pharmaceutical formulations and vaccines may be for administrationby oral (solid or liquid), parenteral (intramuscular, intraperitoneal,intravenous (IV) or subcutaneous injection), transdermal (eitherpassively or using ionophoresis or electroporation), transmucosal(nasal, vaginal, rectal, or sublingual), or inhalation routes ofadministration, or using bioerodible inserts and can be formulated indosage forms appropriate for each route of administration.

For targeting a tumor cell in situ, the compositions described above maybe administered to animals including human beings in any suitableformulation. For example, compositions for targeting a tumor cell may beformulated in pharmaceutically acceptable carriers or diluents such asphysiological saline or a buffered salt solution. Suitable carriers anddiluents can be selected on the basis of mode and route ofadministration and standard pharmaceutical practice. A description ofexemplary pharmaceutically acceptable carriers and diluents, as well aspharmaceutical formulations, can be found in Remington's PharmaceuticalSciences, a standard text in this field, and in USP/NF. Other substancesmay be added to the compositions to stabilize and/or preserve thecompositions.

The compositions of the invention may be administered to animals by anyconventional technique. The compositions may be administered directly toa target site by, for example, surgical delivery to an internal orexternal target site, or by catheter to a site accessible by a bloodvessel. Other methods of delivery, e.g., liposomal delivery or diffusionfrom a device impregnated with the composition, are known in the art.The compositions may be administered in a single bolus, multipleinjections, or by continuous infusion (e.g., intravenously). Forparenteral administration, the compositions are preferably formulated ina sterilized pyrogen-free form.

In some embodiments, the compositions or vaccines are administered bypulmonary delivery. The composition or vaccine is delivered to the lungsof a mammal while inhaling and traverses across the lung epitheliallining to the blood stream [see, e.g., Adjei, et al. PharmaceuticalResearch 1990; 7:565 569; Adjei, et al. Int. J. Pharmaceutics 1990;63:135 144 (leuprolide acetate); Braquet, et al. J. CardiovascularPharmacology 1989; 13(sup5):143 146 (endothelin-1); Hubbard, et al.(1989) Annals of Internal Medicine, Vol. III, pp. 206 212 (α1antitrypsin); Smith, et al. J. Clin. Invest. 1989; 84:1145-1146(α1-proteinase); Oswein, et al. “Aerosolization of Proteins”, 1990;Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colo.(recombinant human growth hormone); Debs, et al. J. Immunol. 1988;140:3482 3488 (interferon γ and tumor necrosis factor α); and U.S. Pat.No. 5,284,656 to Platz, et al. (granulocyte colony stimulating factor).A method and composition for pulmonary delivery of drugs for systemiceffect is described in U.S. Pat. No. 5,451,569 to Wong, et al. See alsoU.S. Pat. No. 6,651,655 to Licalsi et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer(Marquest Medical Products, Englewood, Colo.); the Ventolin metered doseinhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhalerpowder inhaler (Fisons Corp., Bedford, Mass.). All such devices requirethe use of formulations suitable for the dispensing of the therapeuticagent. Typically, each formulation is specific to the type of deviceemployed and may involve the use of an appropriate propellant material,in addition to the usual diluents, adjuvants, surfactants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder containing the therapeutic agentsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2 tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the therapeutic agent, and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50 to 90% by weight of the formulation. The therapeutic agentshould most advantageously be prepared in particulate form with anaverage particle size of less than 10 mm (or microns), most preferably0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal or other mucosal delivery of the therapeutic agent is alsocontemplated. Nasal delivery allows the passage to the blood streamdirectly after administering the composition to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran and saponin asan adjuvant.

The composition or vaccine of the present invention may be administeredin conjunction with one or more additional active ingredients,pharmaceutical compositions, or vaccines. The therapeutic agents of thepresent invention may be administered to an animal, preferably a mammal,most preferably a human.

Extended Release Systems:

A first extended release system includes matrix systems, in which theagent is embedded or dispersed in a matrix of another material thatserves to retard the release of the agent into an aqueous environment(i.e., the luminal fluid of the GI tract). When the agent is dispersedin a matrix of this sort, release of the drug takes place principallyfrom the surface of the matrix. Thus the drug is released from thesurface of a device, which incorporates the matrix after it diffusesthrough the matrix or when the surface of the device erodes, exposingthe drug. In some embodiments, both mechanisms can operatesimultaneously. The matrix systems may be large, i.e., tablet sized(about 1 cm), or small (<0.3 cm). The system may be unitary (e.g., abolus), may be divided by virtue of being composed of several sub-units(for example, several capsules which constitute a single dose) which areadministered substantially simultaneously, or may comprise a pluralityof particles, also denoted a multiparticulate. A multiparticulate canhave numerous formulation applications. For example, a multiparticulatemay be used as a powder for filling a capsule shell, or used per se formixing with food to ease the intake.

In a specific embodiment, a matrix multiparticulate, comprises aplurality of the agent-containing particles, each particle comprisingthe agent and/or an analogue thereof e.g. in the form of a solidsolution/dispersion with one or more excipients selected to form amatrix capable of controlling the dissolution rate of the agent into anaqueous medium. The matrix materials useful for this embodiment aregenerally hydrophobic materials such as waxes, some cellulosederivatives, or other hydrophobic polymers. If needed, the matrixmaterials may optionally be formulated with hydrophobic materials, whichcan be used as binders or as enhancers. Matrix materials useful for themanufacture of these dosage forms such as: ethylcellulose, waxes such asparaffin, modified vegetable oils, camauba wax, hydrogenated castor oil,beeswax, and the like, as well as synthetic polymers such as poly(vinylchloride), poly(vinyl acetate), copolymers of vinyl acetate andethylene, polystyrene, and the like. Water soluble or hydrophilicbinders or release modifying agents which can optionally be formulatedinto the matrix include hydrophilic polymers such as hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylcellulose, poly (N-vinyl-2-pyrrolidinone) (PVP), poly(ethylene oxide)(PEO), poly(vinyl alcohol) (PVA), xanthan gum, carrageenan, and othersuch natural and synthetic materials. In addition, materials, whichfunction as release-modifying agents include water-soluble materialssuch as sugars or salts. Preferred water-soluble materials includelactose, sucrose, glucose, and mannitol, as well as hydrophilic polymerslike e.g. HPC, HPMC, and PVP.

In a specific embodiment, a multiparticulate product is defined as beingprocessed by controlled agglomeration. In this case the agent isdissolved or partly dissolved in a suitable meltable carrier and sprayedon carrier particles comprising the matrix substance.

Dose:

An effective dose of a composition of the presently disclosed subjectmatter is administered to a subject in need thereof. A “treatmenteffective amount” or a “therapeutic amount” is an amount of atherapeutic composition sufficient to produce a measurable response(e.g., a biologically or clinically relevant response in a subject beingtreated). Actual dosage levels of active ingredients in the compositionsof the presently disclosed subject matter can be varied so as toadminister an amount of the active compound(s) that is effective toachieve the desired therapeutic response for a particular subject. Theselected dosage level will depend upon the activity of the therapeuticcomposition, the route of administration, combination with other drugsor treatments, the severity of the condition being treated, and thecondition and prior medical history of the subject being treated.However, it is within the skill of the art to start doses of thecompound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. The potency of a composition can vary, andtherefore a “treatment effective amount” can vary. However, using theassay methods described herein, one skilled in the art can readilyassess the potency and efficacy of a candidate compound of the presentlydisclosed subject matter and adjust the therapeutic regimen accordingly.

After review of the disclosure of the presently disclosed subject matterpresented herein, one of ordinary skill in the art can tailor thedosages to an individual subject, taking into account the particularformulation, method of administration to be used with the composition,and particular disease treated. Further calculations of dose canconsider subject height and weight, severity and stage of symptoms, andthe presence of additional deleterious physical conditions. Suchadjustments or variations, as well as evaluation of when and how to makesuch adjustments or variations, are well known to those of ordinaryskill in the art of medicine.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1: Therapeutic Treg Expansion In Vivo by TNFRSF25 PreventsAllergic Lung Inflammation

The tumor necrosis factor superfamily (TNFSF) consists of at least 19ligands and 30 receptors (TNFRSF) that are differentially expressed byboth lymphoid and non-lymphoid cells. In CD3⁺ T cells, TNFSF signalsfunction usually in TCR-dependent ways to support various phases of animmune response including polarization, expansion, effector function,contraction, memory and death. TNFRSF25 (DR3, hereafter referred to asTNFR25) is one of the more recently discovered TNFSF members and isexpressed primarily by CD4⁺ and CD8⁺ T and natural killer T (NKT) cells(Fang, L., Adkins, B., Deyev, V., and Podack, E. R. 2008. J Exp Med205:1037-1048). TNFSF15 (TL1A), the ligand for TNFR25, is expressed bysome endothelial cells and is rapidly induced on dendritic cells andmacrophage/monocytes following TLR4 or FcγR signaling (Meylan, F., etal. 2008. Immunity 29:79-89; Prehn, J. L., et al. 2007. J Immunol178:4033-4038). In vitro studies demonstrate that TNFR25 signaling onCD4⁺, CD8⁺ or natural killer T cells increases IL-2, IL-4 and IFNγproduction subsequent to TCR activation or costimulation by IL-12 andIL-18 (Papadakis, K. A., et al. 2005. J Immunol 174:4985-4990). TNFR25signaling also lowers the threshold of CD4⁺ T cells to TCR inducedproliferation in the absence of CD28 costimulation by an IL-2 dependentmechanism (Meylan et al. 2008; Migone, T. S., et al. 2002. Immunity16:479-492).

Activation of TNFR25 by TL1A exacerbates disease pathology inexperimental asthma, inflammatory bowel disease (IBD), rheumatoidarthritis (RA) and experimental autoimmune encephalomyelitis (EAE)(Pappu, B. P., et al. 2008. J Exp Med 205:1049-1062). In each of thesestudies, antigen dependent TNFR25 stimulation of Th1, Th2 or Th17polarized and TCR activated effector T cells enhances the production ofthe relevant effector cytokines from each T helper subset. TNFR25signals are not required for the differentiation of naïve CD4⁺ T cellstoward Th1, Th2 or Th17 lineages. In several of these reports, mousemodels with genetic ablation of TNFR25 or TL1A (Pappu, B. P. et al.,2008; Takedatsu, H., et al. Gastroenterology 135:552-567. Bull, M. J.,et al. 2008. J Exp Med 205:2457-2464) transgenic mouse models expressinga dominant negative TNFR25 or systemic antibody blockade of TL1A werestudied. No immune abnormalities or disease susceptibilities have beenobserved in mouse models deficient in TL1A or TNFR25 or inautoaggressive disease models where the normal signaling of TL1A toTNFR25 is inhibited. Furthermore, in each of these reports expression ofTNFR25 or TL1A produces a pro-inflammatory phenotype that appears morehazardous to the animal than in the absence of TNFR25 or TL1A. To datethere have been no reports examining the role of TNFR25 on CD4⁺FoxP3⁺regulatory T cells (Treg), although Treg may express TNFR25 (Pappu, B.P., et al. 2008. J Exp Med 205:1049-1062). Given the importance of Tregin preventing lethal autoimmunity), expression of TNFR25 by Treg andfunction of TNFR25 in the pathogenesis of multiple autoaggressivedisease models we decided to study the role of TNFR25 on the function ofTreg. This investigation revealed that TNFR25 is highly expressed byTreg but not FoxP3⁻ CD4⁺ conventional T cells (Tconv). In vivostimulation of TNFR25 in the absence of exogenous antigen using anagonistic antibody, clone 4C12, leads to the rapid and selectiveproliferation of natural Treg, but not Tconv, to 30-35% of all CD4⁺ Tcells within four days of 4C12 treatment and is dependent upon TCRengagement with MHC II and IL-2 signaling. Treg expansion by TNFR25protects against lung inflammation upon airway antigen challenge ofsensitized mice. These data demonstrate a novel role for TNFR25 as aregulator of Treg. This role can protect from disease pathogenesis inallergic asthma. Furthermore, in vivo expansion of natural Treg withTNFR25 agonists would provide a translatable method, as an alternativeto IL-2- or ex vivo-based approaches, to facilitate the clinical use ofTreg therapy in humans.

Materials and Methods:

Mice: Wild type C57BL/6 mice were purchased from Charles RiverLaboratories (Wilmington, Mass.). Foxp3-RFP reporter mice on a B6background (Wan, Y. Y., and Flavell, R. A. 2005. Proc Natl Acad Sci USA102:5126-5131)), FoxP3-GFP (Fontenot, J. D., et al. 2005. Immunity22:329-341; Fontenot, J. D., Gavin, M A, and Rudensky, A. Y. 2003. NatImmunol 4:330-336) and CD45.1 SJL, MHC II^(−/−), IL-2 receptor betamutant, CD80/86^(−/−) and CD4^(−/−) mice were bred in our animalfacility. TL1A^(−/−) mice were purchased from Lexicon Genetics Inc. (TheWoodlands, Tex.) and back-crossed into a C57BL/6 background by SpeedCongenics. Mice were used at 6-12 weeks of age and were maintained inpathogen-free conditions at the UM Animal facilities. All animal useprocedures were approved by the University of Miami Animal Care and UseCommittee.

Antibodies and Reagents.

Commercial antibodies for use in flow cytometry were purchased from BDPharmingen or eBioscience. The Armenian Hamster IgG Isotype control wasbought from eBioscience. DTA-1 (α-GITR) was obtained from BioXCell,LG.3A10 (α-IL-27) from BioLegend and 158321 (α-4-1BB) from R&D Systems.Recombinant mouse IL-2 and anti-IL-2 monoclonal antibody, cloneJES6-1A12, were purchased from eBioscience. Recombinant mouseIL-2/anti-IL-2 complex (IAC) was generated by incubating 10,000 unitsrmIL-2 with 5 μg JES6-1A12 for 15 minutes at 25° C. Armenian hamsterhybridomas producing antibodies to mouse TNFR25 (4C12, agonistic) weregenerated as described previously (Fang, L., Adkins, B., Deyev, V., andPodack, E. R. 2008. J Exp Med 205:1037-1048). 4C12 (α-TNFR25) and OX-86(α-OX40) were produced in hollow fiber bioreactors (Fibercell Systems,Frederick, Md.) and purified from serum-free supernatants on a protein Gcolumn (GE Healthcare, UK). Rapamycin (Rapamune, Wyeth) was used at 75μg/kg/day as previously described (Araki, K., et al. 2009. Nature460:108-112.). Cyclosporin-A (25 mg/kg/day) FK506 (3 mg/kg/day) and Aktinhibitor V (Tricirbine, 1.5 mg/kg/day, or twice per day as indicated)were purchased from Calbiochem/EMD and administered by intraperitonealinjection.

Flow Cytometry and Cell Sorting:

Single cell suspensions were prepared from spleen and lymph nodes. 10⁶cells were pre-blocked with anti-mouse CD16/CD32 and stained withdifferent antibody combinations. Intracellular staining was performedaccording to standard procedures. Flow cytometric analysis was performedon a Becton Dickinson FACS LSR II instrument and DIVA or FlowJosoftware. Cell sorting was done using a FACSAria cell sorter (BD) afterenrichment of splenocytes for CD4⁺ T cells using the EasySep Mouse CD4⁺T cell Pre-Enrichment Kit from Stem Cell Technologies.

Real-Time RT-PCR:

Total RNA was extracted from flash-frozen colonic or lung tissuesections and reverse transcribed using the RNeasy Mini Kit and theQuantiTect Reverse Transcription Kit from QIAGEN, respectively.Real-time PCR was performed in duplicates on an ABI 7300 Light Cyclerusing TaqMan probes from Applied Biosystems. Samples were normalized toβ-actin.

Adoptive Transfer:

For studies in FIGS. 2A and 2B, total CD4⁺ cells were FACS sorted fromFIR mice and the percentage of FoxP3⁺RFP⁺ cells was determined aftersorting. Total CD4+ cells containing 10⁶ FoxP3⁺ cells were adoptivelytransferred (i.v.) into MHC or CD4^(−/−) mice on day-2. On day 0, micewere treated with 4C12 antibody or isotype control. For studies in FIGS.11A-11E, 2×10⁶ FACS sorted CD4⁺FoxP3⁻ or CD4⁺FoxP3⁺ cells from CD45.2⁺FIR mice were adoptively transferred via intravenous injection intoCD45.1 congenic SJL mice. One day later 10 μg of 4C12 was administeredby intraperitoneal injection. The expansion of transferred cells wasfollowed by FACS daily (starting after 3 days) in peripheral bloodcells.

In Vitro Suppression Assays:

1×10⁵ of CD4⁺CD25-cells were plated in 96-well round-bottom plates andactivated with 2 μg soluble anti-CD3 (2C11) antibody in the presence orabsence of APCs (ratio 1:1) and CD4⁺FIR⁺ regulatory T cells at differentratios. Control IgG, 4C12, DTA1 antibodies were added where indicated ata concentration of 10 μg/ml. Cultures were incubated for 72 h and pulsedwith ³H-thymidine (1 μCi/well; Perkin Elmer, Waltham, Mass.) for thelast 6 h. Incorporated isotope was measured by liquid scintillationcounting (Micro Beta TriLux counter, Perkin Elmer).

Allergic Asthma Induction:

Mice were sensitized by i.p. injection of 66 μg ovalbumin (crystallizedchicken egg albumin, grade V; Sigma-Aldrich, St. Louis, Mo.) adsorbed to6.6 mg alum (aluminum potassium sulfate; Sigma-Aldrich) in 200 μl PBS onday 0, with a i.p. boost on day 5. On day 12, mice were injected i.p.with either 20 μg anti-TNFRSF25 agonistic antibody (4C12) or 20 μg goatanti-hamster IgG isotype control (Jackson ImmunoResearch LaboratoriesInc., Westgrove, Pa.) in 200 μl PBS. On day 16, mice were aerosolchallenged with 0.5% ovalbumin (Sigma-Aldrich) in PBS for 1 hour using aBANG nebulizer (CH Technologies, Westwood, N.J.) into a Jaeger-NYUNose-Only Directed-Flow Inhalation Exposure System (CH Technologies). Onday 19, mice were sacrificed, lung perfused with PBS and bronchoalveolarlavages obtained. Lung lobes processed for RNA or for single cellsuspensions made from lung homogenate for flow cytometry analysis, orfor lung histology. Draining bronchial lymph nodes were also procuredfor subsequent RNA analysis as well as flow cytometry analysis.Quantification of periodic acid-Schiff (PAS) stained lung sections wasperformed using MacBiophotonics Image J software by color deconvolution(using the H PAS vector) followed by thresholding of images (color [2],set to 100) and counted using the nucleus counter (limits set to between100-1000).

Statistical Analysis:

All graphing and statistical analysis were performed using the ABI Prismprogram. Paired analysis was performed using the students T test.Multiple variable analysis was performed using one-way ANOVA and Tukeypost-test. Significance is indicated as * (p<0.05), ** (p<0.01) and ***(p<0.001).

Results

TNFR25 is Highly Expressed by Regulatory T Cells:

Prior to this study, there have been no reports demonstrating a functionfor TNFR25 on CD4⁺FoxP3⁺ regulatory T cells (Treg). To confirm whetherthere was expression of TNFR25 by Treg, FoxP3⁻CD4⁺ (Tconv) and Treg weresingle-cell sorted from FoxP3 reporter mice to over 99% purity andsubsequently analyzed by flow cytometry for expression of TNFR25 as wellas GITR (TNFRSF18), OX40 (TNFRSF4, CD134) and 4-1BB (TNFRSF9, CD137).Sorting of live Treg was made possible by use of FoxP3-reporter mice(FIR mice) expressing a red fluorescent protein knock-in transgene froma bicistronic construct under the FoxP3 promoter. This analysis revealedthat while TNFR25, OX40, GITR and 4-1BB are all expressed by both Tregand Tconv, the greatest relative difference in expression levels wasobserved by very high expression of TNFR25 in Treg compared to lowexpression by Tconv (FIG. 1A). Without wishing to be bound by theory,the differential expression of TNFR25 between Treg and Tconv indicatedthat TNFR25 may play an important role in the function of Treg.

TNFR25 Stimulation Rapidly Expands Treg In Vivo:

The generation of a TNFR25 agonistic antibody, clone 4C12 was describedpreviously (Fang, L., Adkins, B., Deyev, V., and Podack, E. R. 2008. JExp Med 205:1037-1048). By use of FIR mice the frequency and phenotypeof the Treg population was continuously monitored in peripheral bloodfollowing treatment with the TNFR25 agonistic antibody, 4C12.Intraperitoneal (i.p) injection of 4C12 induced rapid and highlyreproducible expansion of CD4⁺FoxP3⁺ Tregs in vivo (FIG. 1B). Thisexpansion was maximal at 4 and 5 days post 4C12 injection, with FoxP3⁺Tregs comprising 30-35% of the total CD4⁺ T cells in the peripheralblood at the peak of the response. 4C12 expanded Tregs persisted in theperipheral blood and all tissue sites examined for two weeks whileslowly contracting to unstimulated levels. The site of injection did notplay a role in this expansion, as demonstrated by equivalent Tregexpansion following 4C12 injection either intraperitoneally,subcutaneously or intravenously. Treg expansion following 4C12 injectionwas dose-dependent with maximal responses seen with a dose of only 10μg, corresponding to approximately 0.4 mg/kg body weight (FIG. 1B).Treatment of FIR mice with purified mouse TL1A-Ig fusion protein (100μg) was found to induce Treg expansion with a similar magnitude andkinetic as treatment with the 4C12 antibody. Detection of RFP expressionin FIR mice faithfully reports the presence of FoxP3 transcripts,however the possibility exists that it may not guarantee expression ofFoxP3 protein because FoxP3 and RFP are independently translated fromthe FoxP3-RFP transcript. Therefore, expansion of CD4⁺FoxP3⁺ cellsfollowing 4C12 administration was confirmed in wild-type mice bystaining with FoxP3 antibodies and in FoxP3-GFP knock-in reporter micethat express a FoxP3-GFP fusion protein.

Among Treg Expressed TNFR-Members TNFR25 is Unique in Causing TregExpansion:

The TNFRSF members GITR and OX40 are expressed by Treg (FIG. 1A) andeffect Treg activity and proliferation. It is thought that stimulationof Treg by 4-1BB can Modulate both the activity and proliferation ofthese cells. Furthermore, stimulation of CD4⁺FoxP3⁻ cells via TNFRSFmember CD27 are thought to induce FoxP3 expression. Given the overlapbetween either functional suppression or induction of Tregs betweenTNFR25 and these other TNFSF members, Treg expansion was compared invivo after stimulation of TNFR25, OX40, 4-1BB, GITR or CD27. In allcases well characterized agonistic monoclonal antibodies to therespective receptor were used to trigger specific signaling. Thesestudies demonstrated that TNFR25 is unique among the TNFRSF membersexamined in its ability to selectively induce expansion of Tregs (FIG.1C). It was recently reported that OX40-induced Treg expansion requireddepletion of IL-4, IL-6 and IFNγ (Ruby, C. E., et al. 2009. J Immunol183:4853-4857). In contrast, TNFR25 induced Treg expansion in vivorequired no additional manipulations.

MHC II and IL-2 Signals are Required for TNFR25 Induced TregProliferation:

In vitro, Treg proliferation can be induced with various combinations ofTCR-stimulating antibodies, antigen presenting cells and IL-2 signals.Induction of Treg proliferation in vitro was attempted in these studiesusing many different combinations of anti-CD3 and anti-CD28 antibodies,recombinant 1L-2, TGF-β and retinoic acid with or without TNFR25agonistic antibody, and in all cases TNFR25 stimulation failed toenhance Treg proliferation in vitro, indicating that additional signalswere required (Table 1).

TABLE 1 Conditions tested in vitro using various purified lymphocytepopulations (indicated) to examine requirements for TNFR25 induced Tregproliferation. Condition Splenocytes CD4+ LN 1. Unstim. X X X 2.Unstim. + 4C12 X X X 3. Unstim + 4C12 crossl. X 4. α-CD3 X X X 5.α-CD3 + 4C12 X X X 6. α-CD3 + 4C12 crossl. X 7. α-CD3 + TGF-β X X X 8.α-CD3 + TGF-β + 4C12 X X X 9. α-CD3 + RA X 10. α-CD3 + RA + 4C12 X 11.α-CD3 + RA + 4C12 crossl. X 12. α-CD3 + α-CD28 X X X 13. α-CD3 +α-CD28 + 4C12 X X X 14. α-CD3 + IL-2 X X X 15. α-CD3 + IL-2 + 4C12 X X x16. α-CD3 + α-CD28 + IL-2 X 17. α-CD3 + α-CD28 + IL-2 + 4C12 X 18.α-CD3 + α-CD28 + TGF-β X 19. α-CD3 + α-CD28 + TGF-β + 4C12 X 20. α-CD3 +IL-2 + TGF-β X 21. α-CD3 + IL-2 + TGF-β + 4C12 X 22. α-CD3 + α-CD28 +IL-2 + TGF-β X 23. α-CD3 + α-CD28 + IL-2 + TGF-β + X 4C12 24. One day invivo + 4 days in vitro: x IL-2 + 4C12 titration

Since TNFR25 may influence the responsiveness of CD4⁺ T cells to TCRsignals, the next experiments conducted were to determine whether TNFR25induced Treg proliferation was dependent upon TCR signaling in vivo. MHCor CD4^(−/−) mice were adoptively transferred with total CD4⁺ cellscontaining 10⁶ CD4⁺FoxP3⁺ cells purified from FIR mice. Because MHCII^(−/−) mice are deficient in CD4⁺ T cells, it was decided to useCD4^(−/−) mice as a control population to control for any homeostaticexpansion that may occur following adoptive transfer into a CD4⁺ T celldepleted environment. Mice were treated with 4C12 or isotype controlantibody 2 days after adoptive transfer and the percentage and absolutenumbers of Treg were determined at days 4 and 6 after antibody injection(FIGS. 2A, 2B). These data demonstrate that although Treg expand to asimilar degree in wild-type and CD4^(−/−) mice, MHC II molecules arerequired for TNFR25 induced Treg proliferation in vivo. The percentageof adoptively transferred Treg in MHC II^(−/−) mice was observed to belower than in CD4^(−/−) mice because MHC II^(−/−) mice have a greaternumber of CD4⁺ cells at baseline than CD4−/− mice (FIG. 2A). Acomparison of the absolute numbers of adoptively transferred Treghowever (FIG. 2B), indicates that equivalent absolute numbers of Tregwere recovered from the two groups. These studies demonstrate arequirement for MHC II signals in TNFR25 induced Treg proliferation,which indirectly implies that TCR signaling is required for Treg tobecome permissive to TNFR25 signaling, similar to TNFR25 signaling inTconv cells. To provide additional evidence that TCR signals arerequired for TNFR25 induced Treg proliferation, mice were pre-treatedwith cyclosporine A or FK506 and Treg numbers were analyzed subsequentto treatment with the 4C12 or isotype control antibodies (FIGS. 2C, 2D).These studies demonstrate that, similar to what was observed in theabsence of MHC II signals; TNFR25 triggering in the presence ofcyclosporine A or FK506 fails to induce Treg proliferation. Therequirement for cognate self-antigen in the MHC II is under furtherinvestigation, but such a requirement may provide additional explanationfor Treg selectivity of TNFR25 (in addition to the selective expressionof TNFR25 on Treg, FIGS. 8A-8C) in the absence of exogenous antigen.

It thought that TNFR25 signaling increases the responsiveness of Tconvto IL-2 signals subsequent to TCR signals in the absence of CD28costimulation. Given the requirement for both MHCII and NFAT activationfor TNFR25 induced proliferation of Treg (FIGS. 2A-2D) it was determinedwhether IL-2 or CD80/86 signals were additionally required. Tregexpansion in mice expressing a non-functional 1L-2 receptor beta chain(FIG. 2E) and CD80/86^(−/−) mice (FIG. 2F) was determined 4 days afterinjection of 4C12. These data demonstrate that TCR and IL-2 receptorsignaling, but not CD80 or CD86 costimulation is required for TNFR25induced Treg expansion in vivo. Without wishing to be bound by theory,CD28 and CTLA-4 signaling in Treg may not be a requirement for TNFR25induced proliferation. Furthermore, because combined TNFR25, TCRstimulation and IL-2 signaling fail to induce Treg proliferation invitro, additional signals are required that are also underinvestigation.

TNFR25 Stimulated Treg are Hyper-Responsive to IL-2 InducedProliferation Ex Vivo:

Although the requirements for TNFR25 induced Treg proliferation in vitroneed further study, it was observed that Treg purified from mice treatedwith TNFR25 agonistic antibodies were hyper-responsive to IL-2 inducedproliferation ex vivo (FIG. 3A). These data corroborate the importanceof IL-2 signals in TNFR25 induced Treg expansion (FIG. 2E), and indicatethat TNFR25 triggering induces Treg expansion by influencing thesensitivity of Treg to IL-2 signals. Several potential mechanisms wereenvisioned, that could explain this observation: 1) TNFR25 couldincrease the expression of IL-2 receptor subunits on Treg, 2) TNFR25could enhance STAT5 activation in Treg, 3) TNFR25 could enhance mTORactivation in Treg and 4) TNFR25 could enhance PI3-kinase/Akt activationin Treg. To determine the expression of the IL-2 receptor alpha, betaand gamma chains, flow cytometry was performed on Treg undergoingexpansion in vivo subsequent to treatment with the 4C12 antibody ascompared to Treg isolated from mice treated with IgG control antibodies(FIG. 3B). These data demonstrate that while the expression of the IL-2receptor alpha chain (CD25) actually decreases following exposure to4C12 (FIG. 7A), expression of the beta and gamma chains (CD122 andCD132, respectively) remain unchanged on Treg isolated from mice treatedwith 4C12 and isotype control antibodies (FIG. 3B), effectivelyeliminating option (I) as a possibility. To determine whetherphosphorylation of STAT5 was enhanced in 4C12 treated mice, Treg wereisolated from mice treated 4 days previously with 4C12 or isotypecontrol antibody and exposed to IL-2 ex vivo (10 ng/ml, 15 min).Subsequent staining of these Treg with phospho-specific antibodiesdemonstrated that neither STAT5 nor S6 phosphorylation were enhanced inTreg isolated from 4C12 treated mice as compared to control mice,effectively eliminating the second possibility (FIG. 3C). Subsequently,TNFR25 induced Treg proliferation in vivo was found to be unchanged inthe presence of the mTOR inhibitor, rapamycin, eliminating the thirdpossibility (FIG. 3D). Finally, to determine whether Akt signaling wasrequired for TNFR25 induced Treg proliferation, mice were treated withTNFR25 agonistic antibodies or control antibody in the presence orabsence of the Akt1/2/3 selective inhibitor, tricirbine (Akt inhibitorV, AktiV). These studies demonstrated that selective inhibition of Aktactivation was sufficient to inhibit TNFR25 induced Treg proliferationfrom 33.69±1.253% in vehicle treated controls to 22.43±1.352% (N=6) whentreated once-daily with AktiV (data not shown, p<0.001) and to18.20±2.117% (N=3) when treated twice-daily with AktiV (FIG. 3E,p=0.0003).

Comparison of TNFR25 or IL-2 Antibody Complex Induced Treg Expansion inAntigen Naïve Mice:

The only other agent that selectively expands Treg in vivo was reportedby Boyman et al, (Science 311:1924-1927, 2006) through use of a complexof recombinant IL-2 and a specific anti-IL-2 antibody (IAC), cloneJES6-1A12. Thus, in vivo Treg expansion was directly compared followingtreatment with either 4C12 or IAC (FIG. 7A). This analysis demonstratesthat the magnitude and kinetics of Treg expansion were similar followingtreatment with 4C12 or IAC in vivo. However the contraction of expandedTregs was observed to be prolonged following treatment with 4C12 ascompared to treatment with IAC. In contrast to TNFR25 expanded Treg,which expressed intermediate levels of CD25, IAC expanded Treg wereobserved to express high levels of CD25 (FIG. 7B). No other differencesin expression of CD11a, CD28, CD45RA, CD62L, CD127, intra- orextra-cellular CTLA-4, OX40, PD-1, IL-17A or IFNγ were found bycomparison of Treg expanded by 4C12 to Treg expanded by IAC.

In Vivo Treg Expansion by TNFR25 Reduces Allergic Lung Inflammation:

To determine whether 4C12 expanded Treg prevent inflammation in adisease model, it was tested whether this treatment could reduceinflammation in a well characterized model of allergic lung inflammationinduced in ovalbumin/alum primed mice followed by airway ovalbuminchallenge. Mice were primed with ovalbumin/alum on day 0 and 5 and thentreated with 4C12 or hamster IgG on day 12. Four days later, at the timeof maximal Treg expansion, the airways were challenged with ovalbuminaerosolized in PBS or a PBS saline control. Maximal expansion of Tregswas confirmed by monitoring Treg in the peripheral blood during thisperiod (FIG. 4A). 4C12 induced Treg expansion following ovalbumin/alumsensitization was slightly delayed in the first two days, as compared toexpansion in non-sensitized mice, but Tregs then rapidly expanded to ahigher proportion (50-55%) of total CD4+ T cells by day 4 (FIGS. 4B,4C). Mice were sacrificed three-days after aerosolization and bronchialalveolar lavage fluid (BALF), bronchial lymph nodes (bLN) and lungtissue were analyzed.

The total number of cells isolated from the lungs was unchanged betweencontrol or 4C12 treated animals. Consistent with this observation, thenumber of CD4⁺ and CD8⁺ T cells within the lungs was similar betweencontrol and 4C12 treated mice, however in 4C12 treated mice the numberof Treg was significantly increased (FIG. 4B). Analysis of thecomposition of Treg within the lung tissue revealed that seven daysafter 4C12 administration (and 3 days after aerosolization) thefrequency of Tregs in the lungs remained at 55% of all CD4⁺ T cells ascompared to 22% in hamster IgG treated mice (FIG. 4C). It has beenreported that the balance of Tconv to Treg is a better predictor ofdisease pathogenesis than merely the total number of Treg (Tang, Q., etal. 2008. Immunity 28:687-697; Monteiro, J. P., et al. 2008. J Immunol181:5895-5903); the ratio of CD4⁺FoxP3⁻ (Tconv) to Treg was determinedin lung tissue (Table 2). To confirm that the phenotype oflung-infiltrating Treg was consistent with the phenotype ofTNFR25-expanded Treg in disease-free mice, lung infiltrating Treg wereanalyzed and found to be indistinguishable from lung-infiltrating Tregisolated from IgG treated mice in expression of GITR, OX40, PD-1, CD44,CD62L and CD69.

TABLE 2 The total number of CD4⁺FoxP3⁻ (Tconv), CD4⁺FoxP3⁺ (Treg) andthe ratio of Tconv to Treg cells from total lung cells harvested asdescribed for FIGS. 10A-10E are shown. Cell numbers were cal- culated bymultiplying the number of cells obtained in a single cell suspension ofthe left lung × the percentage of lymphoid gated cells out of totalcells analyzed by flow cytometry × the percentage of Tconv or Treg cellswithin the lymphoid gated cell population. 4C12 decreases absoluteT_(conv) number and T_(conv):T_(reg) ratio IgG 4C12 # of T_(conv)/lung78,200 23,340 # of T_(reg)/lung 10,700 44,400 T_(conv):T_(reg) ratio 7:11:2

Consistent with analysis of lung tissue cells, the total number of cellsisolated from BALF was significantly increased following aerosolchallenge containing ovalbumin, but not saline aerosol control, in allconditions, but was markedly reduced by 4C12 treatment. The total numberof eosinophils within the BALF roughly mirrored the total number of BALFcells, and pre-treatment with 4C12 was observed to significantly reducethe severity of airway eosinophilia (FIG. 4D).

The pro-inflammatory cytokines IL-4, IL-5 and IL-13 have been stronglyimplicated in the pathogenesis of allergic lung inflammation. Todetermine whether expression of these cytokines was reduced bypre-treatment with 4C12, total RNA was extracted from flash-frozen lungsthree days after aerosolization and analyzed by RT-PCR. This analysisdemonstrates that the expression of IL-4, IL-5 and IL-13 amonglung-infiltrating CD4+ cells is significantly reduced followingtreatment with 4C12, but remains elevated following treatment withisotype control antibody as compared to saline-aerosolized controls(FIG. 4E). As an additional control, the level of FoxP3 RNA expressionwas analyzed and mirrored the same relative proportions of FoxP3expressing CD4⁺ cells as seen by flow cytometry (compare FIG. 4C to 4E).Lung tissue histology confirmed these findings, demonstrating reducedlymphocyte infiltration and airway mucus production following 4C12treatment as compared to saline aerosolized controls (FIG. 4F andquantified in FIG. 4G).

TNFR25 Expands Treg without Activating or Expanding Tconv:

To determine the phenotype of the 4C12-expanded Tregs, we analyzedCD4⁺FoxP3⁺ cells isolated from peripheral lymph nodes, mesenteric lymphnodes and spleens from mice that had been injected with 4C12 or IgGisotype control. 4C12 expanded Tregs were predominantly CD4⁺FoxP3⁺CD25intermediate (int) cells and were found to be expanded in all secondarylymphoid organs analyzed (FIGS. 5A, 5B and FIGS. 8A, 8D). 4C12 treatmentdid not alter the expression of CD11a, CD28, CD45RA, CD62L, CD127,intra- or extra-cellular CTLA-4, OX40, PD-1, IL-17A or IFNγ by Treg.Although all CD4⁺FoxP3⁺ cells remained GITR positive following treatmentwith 4C12, the proportion of CD4⁺FoxP3⁺ cells that expressed GITRshifted in favor of the CD25 int subset following 4C12 treatment (FIG.8B). The αEβ7 integrin is expressed by a highly suppressive subset ofCD4⁺FoxP3⁺ that can be either CD25 positive or negative. Analysis ofCD103 expression revealed increased expression of CD103 by 4C12 expandedTreg but not control Treg (FIG. 8C). Importantly, analysis of CD4⁺FoxP3⁻cells and of CD8⁺ cells following treatment with 4C12 revealed thatTNFR25 signaling does not increase the absolute number or proportion ofeither of these cell populations. To determine whether treatment with4C12 stimulated the proliferation of non-Treg cells, CD4⁺ Tconv and CD8⁺T cells were stained with the proliferation marker, Ki67. This analysisillustrated that treatment with 4C12 in the absence of exogenous antigendid not increase Tconv or CD8⁺ T cell proliferation. Moreover, stainingof CD8⁺ cells and FoxP3⁻ CD4⁺ cells for CD44, CD62L and CD69 revealed nodifferences between 4C12 and IgG treated mice. Thus, TNFR25 signalingselectively expands Tregs without inducing expansion or activation ofCD4⁺ or CD8⁺ effector cells in the absence of exogenous antigen.

TNFR25 Stimulation Induces Proliferation of Natural Treg In Vivo:

The increase in Tregs following 4C12 treatment could result either fromde novo FoxP3 expression or from the proliferation of CD4⁺FoxP3⁺ cells.To differentiate between these two possibilities, the expression of theproliferative marker, Ki67, on CD4+FoxP3+ cells was determined (FIGS.5C, 5D). As the data showed that the increase in the ratio ofCD4⁺FoxP3⁺CD25^(int) cells relative to CD4⁺FoxP3⁺CD25^(hi) cells, themajority of Ki67⁺ cells was CD4⁺FoxP3⁺CD25^(int) in mice that weretreated with 4C12. A smaller proportion (˜27%) of CD4⁺FoxP3⁺ cells didnot stain for Ki67 (FIGS. 5C, 5D), and the majority of these cells wereCD25^(hi). It remains unclear whether the observed proliferation ofCD25^(int) cells following treatment with 4C12 resulted from theselective stimulation of CD25^(int) cells or whether Treg werestimulated to proliferate regardless of CD25 expression, which was thenreduced during proliferation.

Increased proliferation by CD4⁺FoxP3⁺CD25 int cells does notconclusively rule-out the possibility that TNFR25 signaling couldstimulate de novo FoxP3 expression by CD4⁺FoxP3⁻ cells. To examine thispossibility adoptive transfer experiments were performed by infusinghighly purified (>99% purity) CD4⁺FoxP3⁻ or CD4⁺FoxP3⁺ cells fromCD45.2⁺ FIR mice into CD45.1 congenic B6/SJL mice. These studies allowedfor the tracking of adoptively transferred CD45.2⁺ cells followingtreatment with 4C12 in CD45.1⁺ hosts and to monitor persistence,induction or silencing of FoxP3-RFP by adoptively transferredCD45.2⁺CD4⁺ cells (FIGS. 5E-5H). It was deliberately chosen to performthese experiments in fully immunocompetent mice to avoid anycomplications that may arise from homeostatic expansion of Tregsfollowing adoptive transfer into genetically or experimentallyimmunodeficient strains. Transfer of 2×10⁶ sorted cells (FIG. 9A) intoimmunocompetent CD45.1⁺ recipients was sufficient to detect a rare, buteasily distinguishable, population of CD45.2⁺CD4⁺ cells in theperipheral blood for at least two weeks post adoptive transfer (FIGS.5G, 5H). TNFR25 stimulation of recipient mice by 4C12 after adoptivetransfer did not stimulate de novo FoxP3 expression by CD4⁺FoxP3⁻ cells(FIG. 5E) which remained at 0.5% frequency and FoxP3−RFP− regardless of4C12 or control antibody treatment. The frequency of FoxP3⁺RFP⁺ cellsafter adoptive transfer of 2×10⁶ cells was 0.04% of the CD4 cells inperipheral blood in mice treated with control antibody and increased to0.11% in 4C12 treated mice (FIG. 5F) a three-fold increase of thefrequency FoxP3⁺RFP⁺CD45.2⁺ cells. This result is consistent with theextent of expansion of FoxP3⁺ Treg by the TNFR25 agonistic antibody innon-transferred mice (FIG. 1B). The data indicate that 4C12 treatmentselectively stimulates the proliferation of CD4⁺FoxP3⁺ cells, whichmaintain FoxP3 expression following expansion (FIG. 5F). The data showthat TNFR25 signaling stimulates primarily increased proliferation ofCD4⁺FoxP3⁺CD25^(int) cells resulting in a systemic increase in Tregs.These studies also demonstrate that while the adoptively transferredCD4⁺FoxP3⁻ cells do not expand at any time following 4C12 treatment(FIG. 5G), the expansion of adoptively transferred CD4⁺FoxP3⁺ cellsfollows similar kinetics as the expansion and contraction of endogenousTregs in FIR mice (compare FIG. 5H to FIG. 1B). Importantly, theadoptively transferred CD4⁺FoxP3⁺ cells maintain FoxP3 expression bothduring and after expansion, suggesting that the observed contraction ofthe expanded Treg pool results from cell death rather than from loss ofFoxP3 expression (FIGS. 5F, 5H). If the expanded pool of adoptivelytransferred CD45.2⁺CD4⁺FoxP3⁺ cells were losing FoxP3 expression at anypoint throughout the course of the experiment, the fraction ofCD4⁺FoxP3⁻ cells within the CD45.2⁺CD4⁺ cells would have increased,however this did not occur. A small proportion of adoptively transferredCD4⁺FoxP3⁻ cells (<5%) were observed to exhibit FoxP3 expression (FIG.5E) and a small proportion of transferred CD4⁺FoxP3⁺ cells (<5%) lostFoxP3 expression (FIG. 5F) over the course of the experiment. Such minorinstabilities in FoxP3 expression likely explain these observations.

TNFR25 Expanded Treg are Highly Suppressive Ex Vivo:

To determine whether 4C12 expanded Tregs retain suppressive activity,Treg cells were purified from FIR mice four days after treatment witheither 4C12 or IgG isotype control antibody (FIG. 8A). These Tregsubsets were then used in a traditional proliferation assay. PurifiedTregs from 4C12 treated mice suppressed proliferation of CD4⁺CD25⁻ cellsto a greater degree than those from isotype control antibody treatedmice (FIGS. 6A-6D). Suppression of Tconv proliferation by 4C12 expandedTreg was observed both in the presence and absence of antigen presentingcells (APC) in the in vitro suppression assay (FIG. 6A vs. 6B). Todetermine whether addition of 4C12 during the suppression assaymodulated the suppressive activity of Treg, identical assays wereperformed as described (FIGS. 6A, 6B) in the presence of 4C12 or aGITR-agonistic antibody known to release Tconv from Treg-mediatedsuppression (clone DTA-1) or isotype control antibody (FIGS. 6C, 6D).The presence of agonistic TNFR25 or GITR antibodies partially restoredTconv proliferation, with both antibodies producing a similar effect inthe absence of APC regardless of whether the Treg were obtained from4C12 or IgG-isotype control treated mice (FIG. 6C). Interestingly, inthe presence of APC, DTA-1 induced the proliferation of Tconv in thepresence of Treg from IgG-isotype control treated mice to a greaterextent than with Treg from 4C12 treated mice (FIG. 6D). The presence ofAPC did not significantly alter the partial restoration of Tconvproliferation in the presence of 4C12. Controls also demonstrated thatthe stimulatory effect of 4C12 on Tconv alone was minimal, andsignificantly less than the stimulatory effect of GITR on Tconv (FIGS.6C, 6D). To further demonstrate that inhibition of Treg suppressiveactivity by 4C12 was specific to the effect of TNFR25 expressed by Tregand not Tconv, suppression assays were performed using transgenic Tconvexpressing a dominant negative TNFR25 (FIG. 6E). These data demonstratethat the inhibition of Treg suppressive activity by TNFR25 signalingoccurs under conditions where only Treg express a functional TNFR25,indicating that this effect is due to signaling by TNFR25 on Treg andnot Tconv. Notably, Tregs expanded in vivo with 4C12 and then subjectedto the in vitro suppression assays (FIGS. 6A-6B) are highly suppressiveunder conditions where the 4C12 antibody is no longer present. It isonly when the 4C12 antibody is, maintained in the course of thesuppression assay that partial inhibition of Treg suppressive activityis observed. Because 4C12 induced the proliferation of CD25^(int) Treg,and in some studies the level of expression of CD25 is predictive of thesuppressive activity of Treg, the suppressive activity of CD25^(hi) andCD25^(int) Treg sorted from mice following treatment with 4C12 orisotype control antibody was compared (FIG. 9B and FIG. 6F). Thesuppressive activity did not depend on the level of CD25 expressionsince CD25^(hi) and CD25^(int) Treg were both highly suppressive in theproliferation assay (FIG. 6F). Interestingly, the 4C12-expandedCD25^(int) Treg had slightly greater suppressive activity than theCD25^(int) Treg from the IgG-treated mice (FIG. 6F, bars 3-4). Thisfinding indicates that the increased suppressive activity of4C12-expanded Treg as compared to IgG-treated Treg (FIGS. 6A-6D) is atleast partially attributable to the activity of CD25^(int) cells.

Discussion

Members of the TNF receptor family have been recognized as importantcostimulators of immune effector cell responses and as inducers ofapoptosis. Here, TNFR25 was identified as a novel non-redundant functionas regulator of T regulatory cells. TNFR25 mediates robust expansion ofTreg in vivo in immune competent mice while at the same time partiallyrestraining their suppressive activity. No other physiological signals,including those of other TNFR-family members have been reported to exertsimilar activity on Tregs. Further, the observation that TNFR25 signalsinduce Treg expansion with a similar magnitude and kinetic to the onlyother reported reagent to selectively expand Treg (IL-2/anti-IL-2antibody complexes (Boyman, O., et al. 2006. Science 311:1924-1927),indicates that TNFR25 agonists may provide a translatable alternative toIL-2 based therapies for therapeutic use in humans.

Without wishing to be bound by theory, the TNFR25 and TL1A receptor:ligand pair is implicated in the generation of pathogenic inflammationin various disease models. Heretofore, there is not a single reportwhich identifies a role for TNFR25 or TL1A in maintaining health orpreventing disease, which indicates that such a role had evadeddiscovery. The availability of the TNFR25 agonistic antibody, 4C12,enabled for the first time the study of TNFR25 on various T-cell subsetsin a setting where the temporal availability of TNFR25 signals,inflammatory signals and exogenous antigen could be independentlycontrolled. The identification of a protective role for TNFR25 expandedTreg in allergic lung inflammation does not contradict previous studiesimplicating TL1A in the exacerbation of allergic lung inflammationbecause in the current studies TNFR25 signaling precedes antigenexposure whereas in previous studies TNFR25 signals follow antigenchallenge. Rather, the differential expression of TNFR25 by Treg (highexpression) as compared to Tconv (low expression) indicates that thesequence of exposure of T cells to antigen, costimulatory signals orTL1A may govern whether a particular inflammatory response is suppressedby Treg or induced by Tconv. In the current studies, treatment withTNFR25 agonists prior to airway antigen challenge induced thepreferential accumulation of Treg, but not Tconv, within the airways andwas associated with a reduction in production of IL-4, IL-5 and IL-13 aswell as reduced eosinophilia and mucus production in thebroncheo-alveolar space.

MHC II and IL-2 signals, but not CD80/86 costimulation, were needed forTNFR25 induced Treg proliferation. Although MHCII and IL-2 signals arerequired for TNFR25 induced Treg proliferation, provision of TCR andIL-2 signals are not sufficient to induce proliferation of Treg invitro, indicating that additional signals may be required which areunder further investigation. Although the requirement for MHCII stronglyimplicates the Treg-expressed TCR in TNFR25 induced Treg proliferation,these data are indirect. As additional evidence for a role of the TCR inthis process, it was observed that TNFR25 triggering could not induceTreg proliferation in the presence of the NFAT inhibitors, cyclosporin-Aor FK506; providing evidence that signaling events downstream of the TCRinfluence Treg proliferation. These data indicate that both Treg andTconv may become permissive to TNFR25 signaling subsequent to TCRligation, and that the Treg-selectivity of TNFR25 agonistic antibodiesmay be at least partially due to the availability of self-antigen undernon-inflammatory conditions. Whether or not persistent TCR stimulationwith self-antigen also contributes to the increased expression of TNFR25in Treg as compared to Tconv, or whether this difference is maintainedby unrelated signaling pathways is also not known.

Given that at least two additional receptor pathways (IL-2 receptor andTCR) are required for TNFR25 triggered Treg proliferation, theconfluence of signaling pathways downstream of these receptors leadingto Treg proliferation may be complex. A clue as to how these pathwaysmay interact was provided by the observation that ex vivo,TNFR25-triggered Treg was hyper-responsive to IL-2 signals. It wassubsequently determined that the PI3-kinase/Akt pathway provided a linkdownstream of the IL-2 receptor that is important for TNFR25 inducedTreg proliferation. These data indicate that PTEN-mediated inhibition ofthe PI3-kinase/Akt pathway restricts the proliferation of Tregdownstream of IL-2 signaling. Identification of MHCII, IL-2R, NFAT andAkt provide a tangible starting point for elucidating the signalingevents downstream of TNFR25 triggering that culminates in Tregproliferation, but additional studies are to be conducted to elucidatethe molecular mechanisms of cross-talk between these various pathways.

TNFR25 induced Treg expansion occurs with a similar kinetic andmagnitude to Treg expansion induced by IAC, but results in an increasein the proportion of CD25^(int) rather than CD25^(hi) cells. Theimportance of this observation is unknown; however the increase in CD25expression by Treg following exposure to IAC suggests apositive-feedback loop driven by the increased availability of IL-2. Inthe case of TNFR25 induced Treg expansion, the concentration of IL-2 isnot manipulated, so the resulting decrease in CD25 expression byproliferating Treg may result from increased competition for endogenousIL-2 from an expanding Treg population. Interrogation of other Tregexpressed surface markers revealed few differences between TNFR25 andIAC expanded Treg, although some, including GITR, fluctuated between theCD25^(int) and CD25^(hi) populations. The only marker analyzed that wasconsistently increased following 4C12 treatment was CD103, whichcontributes to the retention of Tregs within tissues.

The data complement recent data reporting roles for TNFR25 stimulationin the induction of inflammatory responses with the inhibition of Tregsuppressive activity into a unified theory for the role of TL1A:TNFR25interactions in both the induction and resolution of tissueinflammation. The precise mechanism by which TNFR25 stimulation inducesboth the proliferation of Tregs and inhibits their suppressive activityremain unclear, in part because the signaling pathways activated byTNFR25 signals are not well understood, and are under furtherinvestigation. In addition, it is unknown whether TNFR25-inducedexpansion of Treg in vivo is dependent upon the recognition ofself-antigen and, similar to IAC, the conditions necessary forTNFR25-induced Treg expansion in vitro remain unclear and are underfurther investigation. Without wishing to be bound by theory, it ishypothesized that the identification of a requirement in vivo for MHC IIto permit TNFR25 induced Treg proliferation indicates that TCRengagement is a general requirement for TNFR25 induced T cellcostimulation, and that the Treg selectivity of TNFR25 in the absence ofexogenous antigen is maintained both by the preferential expression ofTNFR25 by Treg and by the availability of self-antigen presented by MHCII. The increased responsiveness of Tregs to TNFR25 stimulation fromimmunized versus non-immunized mice is also intriguing, and may indicatedistinct functions for TNFR25 in primary versus secondary immuneresponses.

Regardless of the mechanism, due to the importance of TNFR25 signalingto the pathogenesis of a growing number of inflammatory diseases(asthma, IBD, EAE, RA) it is important to understand the spatio-temporalrole which TNFR25 signaling exerts on various CD4⁺ T cell subsets. It ishighly likely that, similar to OX40, the temporal context of TNFR25signaling may differentially guide inflammatory or regulatory immunity.The unique ability of TNFR25 signals to rapidly expand and transientlyinhibit CD4⁺FoxP3⁺ natural regulatory T cells may have importantconsequences for the treatment of autoimmune disease, chronic infection,transplantation and cancer.

Example 2: Therapeutic Treg Expansion In Vivo by TNFRSF25 Delays AcuteRejection of Allogeneic Hearts in a Heterotopic Heart Transplant Model

To study tolerance induction by 4C12 expanded natural Treg a heterotopicheart transplant model was chosen which is well described for tolerancestudies. Hearts from CBA/J mice (H2^(d)) were transplanted into theabdomen of C57BL/6 mice (H2^(b)) on day 0. On day-4, one group of micewas treated with the TNFRSF25 agonistic antibody, clone 4C12, byintraperitoneal injection (20 μg/mouse), with the other treated withhamster IgG isotype control antibody. At the time of transplant Tregexpansion in the blood was confirmed in the 4C12 treated group.Allograft survival was monitored by palpating the heart manually and thepulse was graded on a scale from 0 to 4 (0=no pulse; 1=very mild;2=mild; 3=moderate; 4=strong). Rejection is defined as cessation ofpalpable heart beat. At the time of rejection (=when the heartbeatstopped) the graft was removed, formalin fixed and submitted forpathologic examination. Loss of graft function within 48 h of transplantis considered a technical failure (<5%) and omitted from furtheranalysis.

Example 3: Therapeutic Treg Expansion In Vivo by TNFRSF25 AgonistsProtect From Dextran-Sodium Sulfate Induced Colitis, a Mouse Model ofCrohn's Disease

C57BL/6 mice or TL1A knockout mice were provided with 3% dextran sodiumsulfate (DSS) dissolved in drinking water ad libitum for 7 days. Weightwas monitored daily beginning 4 days prior to provision of DSS(experimental day-4). On day-4, one group of mice was treated with theTNFRSF25 agonistic antibody, clone 4C12, by intraperitoneal injection(20 μg/mouse), with the other treated with hamster IgG isotype controlantibody. Mortality was measured when animals lost ≧20% of starting bodyweight (FIG. 10A). In some experiments, animals were sacrificed atexperimental day 5, and total RNA was prepared using the RNeasy miniprepkit (Qiagen) from flash-frozen, PBS-washed, colonic tissue. RNA wassubsequently reverse transcribed (Quantitect RT, Qiagen) and cDNA wasamplified by real-time PCR using Taqman (Applied Biosystems) probes forthe indicated transcripts (FIG. 10B). Data are shown as the fold changein expression in TL1A knockout mice as compared to C57BL/6 control mice.The percentage body weight loss was monitored and plotted over thecourse of the study in each experimental group (FIG. 10C). Inexperiments where animals were sacrificed on experimental day 5 for RNAisolation, mesenteric lymph nodes were isolated for analysis by flowcytometry for the proportion of CD4⁺ cells expressing the transcriptionfactor FoxP3, indicative of the regulatory T cell pool (FIG. 10D).Finally, reverse transcription was performed using RNA isolated from theindicated treatment groups as described for FIG. 10B and subjected toRT-PCR for the indicated transcripts. Error bars indicate mean±S.E.M.for ≧3 mice per experiment and a minimum of 2 experiments per panel.

These data demonstrate that pre-treatment with a TNFRSF25 agonisticantibody leads to the expansion of FoxP3⁺ regulatory cells within thegut, prevents weight loss and lethal inflammation in the colon, andprevents the expression of inflammatory cytokines including IL-1 betaand IL-6 within colonic tissue. Collectively, these data provideevidence that stimulation of TNFRSF25 can prevent lethal gutinflammation in a mouse model commonly used to mimic inflammationcharacteristic of Crohn's disease in humans.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

1. A method of regulating an immune response in vivo, comprising:administering to a subject in need thereof, an agonist of tumor necrosisfactor superfamily receptor 25 (TNFRSF25); wherein the TNFRSF25 agonistmodulates regulatory T cell (Treg) proliferation.
 2. The method of claim1, wherein the TNFRSF25 agonist stimulates signaling of TNFRSF25 andinduces proliferation of CD4⁺FoxP3⁺ cells in the subject as compared toa normal, healthy control.
 3. The method of claim 1, wherein the Tregcell is identified by expression of markers comprising: TNFR25, OX40,GITR or 4-1BB.
 4. The method of claim 3, wherein the Treg celldifferential express high levels of TNFR25 as compared to conventional Tcells (Tconv; CD4⁺FoxP3⁻).
 5. The method of claim 2, wherein the atleast one agent is administered to a patient in a therapeuticallyeffective dose as determined by the proliferation of Treg CD4⁺FoxP3⁺cells in a patient.
 6. The method of claim 1, wherein the TNFRSF25agonist is administered to a patient in a plurality of therapeuticallyeffective doses.
 7. The method of claim 6, wherein the plurality oftherapeutically effective doses of the at least one agent areadministered to a patient and abrogate suppressive activity of the Tregcells as measured by Treg production of suppressor factors, cellproliferation assays, flow cytometry or immunoassays.
 8. The method ofclaim 1, wherein the TNFRSF25 agonist is an antibody, an aptamer, aligand, a small molecule, a peptide, a protein, an oligonucleotide, apolynucleotide, or an organic or inorganic molecule.
 9. (canceled) 10.The method of claim 8, wherein the TNFRSF25 agonist is an antibodyspecific for TNFRSF25. 11-33. (canceled)